Antigen binding peptides (abtides) from peptide libraries

ABSTRACT

Abtides are provided. Abtides are peptides identified by a two-step process of screening random peptide libraries. In the first step, the target ligand is an antibody or receptor (or derivative thereof). The peptides identified in the first screening step are used as target ligands in the second screening step. The peptides identified in the second screening step are abtides. Abtides possess binding specificities that are similar to the binding specificities of the antibodies or receptors that are used in the first screening step. Abtides may be used in place of antibodies in many assays or therapeutic applications.  
     Abtides binding to polymorphic epithelial mucin (PEM) are provided.  
     Also provided are methods of obtaining abtides as well as diagnostic and therapeutic compounds containing abtides.

[0001] This application is a continuation-in-part of co-pending U.S.patent application Ser. No. 08/310,192 filed Sep. 21, 1994, the entirecontents of which are incorporated herein by reference.

1. FIELD OF THE INVENTION

[0002] The present invention relates generally to peptides capable ofspecific binding to ligands of interest. The present invention alsorelates to peptides capable of mimicking the specific binding of areceptor to its ligand, an antibody to its antigen, and the like. Suchpeptides are known as “abtides.” Abtides are identified by first andsecond screening steps of peptide libraries. The first screening stepuses an antibody or receptor as a first target ligand and identifiespeptide sequences (“mimetopes”) which specifically bind to the antibodyor receptor. The mimetopes are then incorporated into a second targetligand in a second screening step to identify abtides that bind themimetope. Abtides mimic the binding specificity of the antibody (to itsantigen) or the receptor (to its ligand) that was used as the firsttarget ligand in the first screening step. The invention further relatesto the use of abtides in the place of antibodies in assays. Theinvention also provides abtide compositions for use in therapy anddiagnosis of disease.

2. BACKGROUND OF THE INVENTION 2.1. Peptide Libraries

[0003] The use of peptide libraries is well known in the art. Suchpeptide libraries have generally been constructed by one of twoapproaches. According to one approach, peptides have been chemicallysynthesized in vitro in several formats. For example, Fodor et al.,1991, Science 251: 767-773, describes use of complex instrumentation,photochemistry and computerized inventory control to synthesize a knownarray of short peptides on an individual microscopic slide. Houghten etal., 1991, Nature 354: 84-86, describes mixtures of free hexapeptides inwhich the first and second residues in each peptide were individuallyand specifically defined. Lam et al., 1991, Nature 354: 82-84, describesa “one bead, one peptide” approach in which a solid phase splitsynthesis scheme produced a library of peptides in which each bead inthe collection had immobilized thereon a single, random sequence ofamino acid residues. For the most part, the chemical synthetic systemshave been directed to generation of arrays of short length peptides,generally fewer than about 10 amino acids or so, more particularly about6-8 amino acids. Direct amino acid sequencing, alone or in combinationwith complex record keeping of the peptide synthesis schemes, isrequired to use these libraries.

[0004] According to a second approach using recombinant DNA techniques,peptides have been expressed in biological systems as either solublefusion proteins or viral capsid fusion proteins.

[0005] A number of peptide libraries according to the second approachhave used the M13 phage. M13 is a filamentous bacteriophage that hasbeen a workhorse in molecular biology laboratories for the past 20years. M13 viral particles consist of six different capsid proteins andone copy of the viral genome, as a single-stranded circular DNAmolecule. Once the M13 DNA has been introduced into a host cell such asE. coli, it is converted into double-stranded, circular DNA. The viralDNA carries a second origin of replication that is used to generate thesingle-stranded DNA found in the viral particles. During viralmorphogenesis, there is an ordered assembly of the single-stranded DNAand the viral proteins, and the viral particles are extruded from cellsin a process much like secretion. The M13 virus is neither lysogenic norlytic like other bacteriophage (e.g., λ); cells, once infected,chronically release virus. This feature leads to high titers of virus ininfected cultures, i.e., 10¹² pfu/ml.

[0006] The genome of the M13 phage is ˜8000 nucleotides in length andhas been completely sequenced. The viral capsid protein, protein III(pIII) is responsible for infection of bacteria. In E. coli, the pillinprotein encoded by the F factor interacts with pIII protein and isresponsible for phage uptake. Hence, all E. coli hosts for M13 virus areconsidered male because they carry the F factor. Several investigatorshave determined from mutational analysis that the 406 amino acid longpIII capsid protein has two domains. The C-terminus anchors the proteinto the viral coat, while portions of the N-terminus of pIII areessential for interaction with the E. coli pillin protein (Crissman andSmith, 1984, Virology 132: 445-455). Although the N-terminus of the pIIIprotein has been shown to be necessary for viral infection, the extremeN-terminus of the mature protein does tolerate alterations. In 1985,George Smith published experiments reporting the use of the pIII proteinof bacteriophage M13 as an experimental system for expressing aheterologous protein on the viral coat surface (Smith, 1985, Science228: 1315-1317). It was later recognized, independently by two groups,that the M13 phage pIII gene display system could be a useful one formapping antibody epitopes. De la Cruz et al., 1988, J. Biol. Chem. 263:4318-4322 cloned and expressed segments of the cDNA encoding thePlasmodium falciparum surface coat protein into the pIII gene, andrecombinant phage were tested for immunoreactivity with a polyclonalantibody. Parmley and Smith, 1988, Gene 73: 305-318 cloned and expressedsegments of the E. coli β-galactosidase gene in the pIII gene andidentified recombinants carrying the epitope of an anti-β-galactosidasemonoclonal antibody. The latter authors also described a process termed“biopanning”, in which mixtures of recombinant phage were incubated withbiotinylated monoclonal antibodies, and phage-antibody complexes couldbe specifically recovered with streptavidin-coated plastic plates.

[0007] In 1989, Parmley and Smith, 1989, Adv. Exp. Med. Biol.251:215-218 suggested that short, synthetic DNA segments cloned into thepIII gene might represent a library of epitopes. These authors reasonedthat since linear epitopes were often ˜6 amino acids in length, itshould be possible to use a random recombinant DNA library to expressall possible hexapeptides to isolate epitopes that bind to antibodies.

[0008] Scott and Smith, 1990, Science 249:386-390 describe constructionand expression of an “epitope library” of hexapeptides on the surface ofM13. The library was made by inserting a 33 base pair Bgl I digestedoligonucleotide sequence into an Sfi I digested phage fd-tet, i.e.,fUSE5 RF. The 33 base pair fragment contains a random or “degenerate”coding sequence (NNK)₆ where N represents G, A, T or C and K representsG or T. The authors stated that the library consisted of 2×10⁸recombinants expressing 4×10⁷ different hexapeptides; theoretically,this library expressed 69% of the 6.4×10⁷ possible peptides (20⁶).Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87: 6378-6382 alsodescribed a somewhat similar library of hexapeptides expressed as pIIIgene fusions of M13 fd phage. PCT publication WO 91/19818 dated Dec. 26,1991 by Dower and Cwirla describes a similar library of pentameric tooctameric random amino acid sequences.

[0009] Devlin et al., 1990, Science, 249:404-406, describes a peptidelibrary of about 15 residues generated using an (NNS) coding scheme foroligonucleotide synthesis in which S is G or C.

[0010] Christian and colleagues have described a phage display library,expressing decapeptides (Christian et al., 1992, J. Mol. Biol.227:711-718). The starting. DNA was generated by means of anoligonucleotide comprising the degenerate codons [NN(G/T)]₁₀ with aself-complementary 3′ terminus. This sequence, in forming a hairpin;creates a self-priming replication site which could be used by T4 DNApolymerase to generate the complementary strand. The double-stranded DNAwas cleaved at the Sfi I sites at the 5′ terminus and hairpin forcloning into the fUSE5 vector described by Scott and Smith, supra.

[0011] Other investigators have used other viral capsid proteins forexpression of non-viral DNA on the surface of phage particles. Theprotein pVIII is a major M13 viral capsid protein and interacts with thesingle stranded DNA of M13 viral particles at its C-terminus. It is 50amino acids long and exists in approximately 2,700 copies per particle.The N-terminus of the protein is exposed and will tolerate insertions,although large inserts have been reported to disrupt the assembly ofpVIII fusion proteins into viral particles (Cesareni, 1992, FEBS Lett.307:66-70). To minimize the negative effect of pVIII fusion proteins, aphagemid system has been utilized. Bacterial cells carrying the phagemidare infected with helper phage and secrete viral particles that have amixture of both wild-type and pVIII fusion capsid molecules. pVIII hasalso served as a site for expressing peptides on the surface of M13viral particles. Four and six amino acid sequences corresponding todifferent segments of the Plasmodium falciparum major surface antigenhave been cloned and expressed in the comparable gene of the filamentousbacteriophage fd (Greenwood et al., 1991, J. Mol. Biol. 220:821-827).

[0012] Lenstra, 1992, J. Immunol. Meth. 152:149-157 describedconstruction of a library by a laborious process encompassing annealingoligonucleotides of about 17 or 23 degenerate bases with an 8 nucleotidelong palindromic sequence at their 3′ ends. This resulted in theexpression of random hexa- or octa-peptides as fusion proteins with theβ-galactosidase protein in a bacterial expression vector. The DNA wasthen converted into a double-stranded form with Klenow DNA polymerase,blunt-end ligated into a vector, and then released as Hind IIIfragments. These fragments were then cloned into an expression vector atthe C-terminus of a truncated β-galactosidase to generate 10⁷recombinants. Colonies were then lysed, blotted on nitrocellulosefilters (10⁴/filter) and screened for immunoreactivity with severaldifferent monoclonal antibodies. A number of clones were isolated byrepeated rounds of screening and were sequenced.

[0013] Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869described a system in which random peptides were fused to the carboxyterminus of the lac repressor. The fusion proteins contained an intactlac amino terminus (which is responsible for specific binding of the lacrepressor to the DNA sequences constituting the lac operator sites). Thenucleotide sequences encoding the fusion protein were cloned into aplasmid containing copies of the lac operator site. Thus, when thefusion protein was expressed in bacteria, it became bound to theoperator sites of the plasmid encoding it. This provided a physicallinkage between the fusion protein and the gene encoding it. Whenbacteria containing the plasmid were screened with ligands for which itwas desired to isolate binding partners, the fusion proteins comprisingpeptides that specifically bound to the ligand were isolated, carryingalong the genes that encoded those fusion proteins.

[0014] A comprehensive reView of various types of peptide libraries canbe found in GaTlop et al., 1994, J. Med. Chem. 37:1233-1251.

2.2. Ligands Used to Screen Peptide Libraries

[0015] Screening of peptide libraries has generally been confined to theuse of a restricted number of ligands. Most commonly, the ligand hasbeen an antibody (Parmley and Smith, 1989, Adv. Exp. Med. Biol.251:215-218; Scott and Smith, 1990, Science 249:386-390). In many cases,the aim of the screening is to identify peptides from the library thatmimic the epitopes to which the antibodies are directed. Thus, given anavailable antibody, peptide libraries are excellent sources foridentifying epitopes or epitope-like molecules of that antibody (Yayonet al., 1993, Proc. Natl. Acad. Sci. USA. 90:10643-10647).

[0016] While previous studies have succeeded in identifying epitopes andepitope-like molecules: from peptide libraries, it has not been realizedin the prior art that this approach could be extended by using theidentified epitopes in a further round of screening of a peptide libraryto identify antibody-like molecules.

[0017] When it has been desired to obtain antibody-like molecules, theprior art has employed peptide libraries that contain naturallyoccurring antibody sequences. This has probably been due to the factthat specific binding by antibodies is known to depend upon a complexstructure involving various complementarity determining regions (CDRs),often from both heavy and light antibody chains. Short peptides wouldnot be expected to mimic such structures and longer peptides werethought to be unsuitable for display in the most commonly usedlibraries.

[0018] McCafferty et al., 1.990, Nature 348:552-554 used PCR to amplifyimmunoglobulin variable (V) region genes and cloned those genes intophage expression vectors. The authors suggested that phage libraries ofV, diversity (D), and joining (J) regions could be screened withantigen. The phage that bound to antigen could then be mutated in theantigen-binding loops of the antibody genes and rescreened. The processcould be repeated several times, ultimately giving rise to phage whichbind the antigen strongly.

[0019] Marks et al., 1991, J. Mol. Biol. 222:581-597 also used PCR toamplify immunoglobulin variable (V) region genes and cloned those genesinto phage expression vectors.

[0020] Kang et al., 1991, Proc., Natl. Acad. Sci. USA 88:4363-4366created a phagemid vector that could be used to express the V andconstant (C) regions of the heavy and light chains of an antibodyspecific for an antigen. The heavy and light chain V-C regions wereengineered to combine in the periplasm to produce an antibody-likemolecule with a functional antigen binding site. Infection of cellsharboring this phagemid with helper phage resulted in the incorporationof the antibody-like molecule on the surface of phage that carried thephagemid DNA. This allowed for identification and enrichment of thesephage by screening with the antigen. It was suggested that the enrichedphage could be subject to mutation and further rounds of screening,leading to the isolation of antibody-like molecules that were capable ofeven stronger binding to the antigen.

[0021] Hoogenboom et al., 1991, Nucleic Acids Res. 19:4133-4137suggested that naive antibody genes might be cloned into phage displaylibraries. This would be followed by random mutation of the clonedantibody genes to generate high affinity variants.

[0022] In the prior art, peptide libraries have been screened withreceptors to identify receptor ligand-like peptides, but peptidelibraries have not been considered useful for identifying suchligand-binding peptides as those that mimic receptors.

[0023] Bass et al., 1990, Proteins: Struct. Func. Genet. 8:309-314 fusedhuman growth hormone (hGH) to the carboxy terminus of the gene IIIprotein of phage fd. This fusion protein was built into a phagemidvector. When cells carrying the phagemid were infected with a helperphage, about 10% of the phage particles produced displayed the fusionprotein on their surfaces. These phage particles were enriched byscreening with hGH receptor-coated beads. It was suggested that thissystem could be used to develop mutants of hGH with altered receptorbinding characteristics.

[0024] Lowman et al., 1991, Biochemistry 30:10832-10838 used an improvedversion of the system of Bass et al. described above to select formutant hGH proteins with exceptionally high affinity for the hGHreceptor. The authors randomly mutagenized the hGH-pIII fusion proteinsat sites near the vicinity of 12 amino acids of hGH that had previouslybeen identified as being important in receptor binding.

[0025] Balass et al., 1993, Proc. Natl. Acad. Sci. USA 90:10638-10642used a phage display library to isolate linear-peptides that mimicked aconformationally dependent epitope of the nicotinic acetylcholinereceptor. This was done by-screening the library with a monoclonalantibody specific for the conformationally dependent epitope. Themonoclonal antibody used was thought to be specific to the acetylcholinereceptor's binding site for its natural ligand, acetylcholine.

[0026] Citation or identification of any reference herein shall not beconstrued as an admission that such reference is available as prior artto the present invention.

3. SUMMARY OF THE INVENTION

[0027] The present invention relates to abtides. As used herein, theterm “abtides” refers to peptides that mimic the binding specificity ofa larger molecule such as an antibody or receptor. Abtides specificallybind to a ligand of interest, in which the ligand is a specific bindingpartner of the larger molecule (e.g. antibody or receptor). To identifythe abtides of the present invention, peptide libraries are screened ina two-step process. The first screening step uses an antibody (orantigen-binding derivative thereof) or receptor (or ligand-bindingderivative thereof) as a first target ligand. This step identifiespeptide sequences termed “epitopes” or “mimetopes” which specificallybind the first target ligand. In the case where an antibody orderivative thereof is used as the first target ligand, a mimetope willoften resemble, either functionally in terms of its binding capabilityand/or structurally in terms of its amino acid sequence, the epitoperecognized by the antibody used as the first ligand. An epitope ormimetope is then used as a second target ligand in a second screeningstep to identify a peptide sequence that specifically binds the epitopeor mimetope. Such peptides are known as “abtides.” Surprisingly, it wasfound by the current inventors and is demonstrated herein that abtidespossess binding specificities strikingly similar to those possessed bythe first target ligands (usually antibodies or receptors) describedabove.

[0028] Abtides are useful since they mimic the binding specificities ofantibodies or receptors. Thus, they may be used in many instances whereantibodies or receptors may be used. The present invention furtherrelates to the use of abtides in the place of antibodies in assays suchas the many types of immunoassays known in the art. Abtides may take theplace of antibodies in such assays as, for example, enzyme-linkedimmunosorbent assays (ELISAs) or sandwich immunoassays. The inventionalso provides abtide compositions for use in therapy and diagnosis. In aspecific example, abtides have been discovered and demonstrated to beuseful in place of antibodies in enzyme-linked immunosorbent assays andin in vivo localization to prostate carcinoma in a xenograft model.

[0029] The use of abtides has many potential advantages over the use ofantibodies or receptors: the smaller size of abtides allows their easierproduction at lower cost, reduced immunogenicity, and may facilitatetheir in vivo delivery if such is desired; biological reactions andfunctions mediated by constant domains of antibodies, and cross-linkingof antibodies/receptors and resulting biological effects can be avoidedif desired.

4. FIGURE LEGENDS

[0030] The present invention may be understood more fully by referenceto the following detailed description of the invention, examples ofspecific embodiments of the invention and the appended figures in which:

[0031]FIG. 1 shows in schematic diagram a general method for identifyingan abtide by a two-step screening process. See Section 5.3 for adiscussion of this method.

[0032]FIG. 2 shows the binding of biotinylated monoclonal antibody7E11-C5 to immobilized mimetope peptide 7E11-9.5. See Section 6.1.2.2for details.

[0033]FIG. 3 shows similarities in the amino acid sequences of the CDR2Land CDR3L regions of monoclonal antibody 7E11-C5 and the 7E11-C5 abtidesof Table 2. The number of amino acids in the abtides that are similar tothe CDRs is indicated in parentheses, along with the percent homology.Dashes indicate gaps which have been added to improve the homology. Inthe case of clones 13 and 16, the homology with CDR2L was greatest ifthe sequence of CDR2L was reversed. The sequence shown for clone 14 isSEQ ID NO: 1; the sequence shown for clone 17 is SEQ ID NO: 2; Thesequence shown for clone 15 is SEQ ID NO: 3; the sequence shown forclone 13 is SEQ ID NO: 4; the sequence shown for clone 16 is SEQ ID NO:5; the sequence shown for CDR3L is SEQ ID NO: 6; the sequence shown forCDR2L is SEQ ID NO: 7; the sequence shown for CDR2L(rev) is SEQ ID NO:8.

[0034]FIG. 4 shows binding of abtides to the 7E11-9.5 mimetope peptidein a dot blot assay as described in Section 6.1.2.1. Numbers along theleft side of the figure refer to the 7E11-C5 abtide that was spotted inthe indicated position. The number 351 refers to the monoclonal antibody7E11-C5, used as a positive control. The numbers along the top of thefigure refer to the various dilutions of the abtide or the monoclonalantibody that were used.

[0035]FIG. 5 shows the binding of biotinylated mimetopes to immobilizedabtides. □ represents binding of mimetope peptideBiotin-LYANPGMYSRLHSPA-NH₂ to 7E11-C5 abtide clone 14; ◯ representsbinding of mimetope peptide Biotin-LYANPGMYSRLHSPA-NH₂ to 7E11-C5 abtideclone 17; ⋄ represents binding of mimetope peptide Biotin-GMYSRLH-NH₂ to7E11-C5 abtide clone 14; Δ represents binding of mimetope peptideBiotin-GMYSRLH-NH₂ to 7E11-C5 abtide clone 17. See Section 6.1.2.2 fordetails.

[0036]FIG. 6 shows the capture of an antigen from a lysate of LNCaPtumor cells by the monoclonal antibody 7E11-C5 and the 7E11-C5 abtideclone 14. See Section 6.1.3 for details.

[0037]FIG. 7 shows the biodistribution of abtide clone 14-DPTA-¹¹¹In inSCID mice bearing human prostate carcinoma LNCaP xenograft tumors 2hours (

, bar on the left for each pair of bars) or 4 hours (

, bar on the right for each pair of bars) post-injection of 2 μg ofpeptide, specific activity 32 μCi/μg. See Section 6.1.4 for details.

[0038]FIG. 8 shows the biodistribution of abtide clone. 17-DPTA-¹¹¹-Inin four SCID mice bearing human prostate LNCaP carcinoma xenografttumors 2 hours (

, leftmost bar for each group of four bars, mouse 1;

second bar from left for each group of four bars, mouse 6) or five hours(

, third bar from left for each group of four bars, mouse 2;

, rightmost bar for each group of four bars, mouse 4) post-injection of0.02 μg of peptide, specific activity 2.4 μCi/ng. See Section 6.1.4 fordetails.

[0039]FIG. 9 shows the biodistribution of ¹¹¹-In labeled controlirrelevant peptide in SCID mice bearing human prostate carcinoma LNCaPxenograft tumors 2 hours (

, leftmost bar for each group of five bars;

, second bar from left for each group of five bars) or 5 hours (

, third bar from left for each group of five bars;

, fourth bar from left for each group of five bars;

, rightmost bar for each group of five bars) post-injection of 1.5 μg ofpeptide, specific activity 30 μCi/μg. See Section 6.1.4 for details.

[0040]FIG. 10 schematically illustrates the construction of the R26 TSARlibrary. The R26 expression library was constructed essentially asdescribed for the TSAR-9 library that is described in PCT publication WO94/18318, dated Aug. 18, 1994; except for the modifications depicted inFIG. 10. The oligonucleotide assembly process depicted in FIG. 10results in expression of peptides with the following amino acidsequence:

[0041] S(S/R)X₁₂πAδX₁₂SR (SEQ ID NO: 89), where π=S, P, T or A; and δ=V,A, D, E OR G.

[0042]FIG. 11 schematically illustrates the construction of the D38 TSARlibrary. The D38 expression library was constructed essentially asdescribed for the TSAR-9 library that is described in PCT publication WO94/18318, dated Aug. 18, 1994, except for the modifications depicted inFIG. 11.

[0043]FIG. 12 schematically illustrates the construction of the DC43TSAR library. The DC43 expression library was constructed essentially asdescribed for the TSAR-9 library that is described in PCT publication WO94/18318, dated Aug. 18, 1994, except for the modifications depicted inFIG. 12.

[0044]FIG. 13 schematically illustrates the oligonucleotides used toconstruct the polymorphic epithelial mucin (PEM) abtide saturationmutagenesis TSAR library (See Section 6.2.2).

5. DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention relates generally to abtides. As usedherein, the term “abtides” refers to peptides that mimic the bindingspecificity of a larger molecule such as an antibody or receptor.Abtides specifically bind to a ligand of interest, in which the ligandis a specific binding partner of the mimicked larger molecule (e.g.antibody or receptor) To identify the abtides of the present invention,peptide libraries are typically screened in a two-step process (see FIG.1). The first screening step uses an antibody (or antigen-bindingderivative thereof) or receptor (or ligand-binding derivative thereof)as a first target ligand. This step identifies peptide sequences termed“epitopes” or “mimetopes” which specifically bind the first targetligand. If the first screening step uses an antibody and the peptideidentified contains the amino acid sequence of the natural antigen thatis responsible for the specific binding of the antigen to the antibody,then the identified peptide is said to be an epitope; if the identifiedpeptide does not contain the sequence of the natural antigen, then theidentified peptide is said to be a mimetope. In the case where anantibody or derivative thereof is used as the first target ligand, amimetope will often resemble, either functionally in terms of itsbinding capability and/or structurally in terms of its amino acidsequence, the epitope recognized by the antibody used as the firstligand.

[0046] A mimetope is then used as a second target ligand in a secondscreening step to identify a peptide sequence that specifically bindsthe epitope or mimetope. Such peptides are known as “abtides.” Abtidespossess binding specificities similar to those possessed by the firsttarget ligands (usually antibodies or receptors) described above.

[0047] In a specific embodiment, the antibody or derivative thereof usedin the first screening step recognizes a tumor antigen, preferably ahuman tumor antigen, most preferably of a malignant tumor.

[0048] The present invention provides a method to successfully screenagainst very small peptide or protein targets, e.g. 5 to 40 amino acids,preferably 10 to 20 amino acids. To date, screening against such targetshas not been successful. The methods of the present invention increasethe likelihood that the abtide obtained will bind its target in acomplex or structurally dependent fashion.

[0049] Abtides are useful since they mimic the binding specificities ofantibodies or receptors. Thus, they may be used in many instances whereantibodies or receptors may be used. The present invention furtherrelates to the use of abtides in the place of antibodies in assays suchas the many types of immunoassays known in the art. Abtides may take thelace of antibodies in such assays as, for example, enzyme-linkedimmunosorbent assays (ELISAs) or sandwich immunoassays. The inventionalso provides abtide compositions for use in therapy and diagnosis ofdisease. In a specific example, abtides have been produced anddemonstrated to be useful in place of antibodies in enzyme-linkedimmunosorbent assays and in in vivo localization to prostate carcinomain xenograft models.

[0050] The use of abtides has many potential advantages over the use ofantibodies or receptors: the smaller size of abtides allows their easierproduction at lower cost, reduced immunogenicity, and facilitates theirin vivo delivery if such is desired; biological reactions and functionsmediated by constant domains of antibodies, and cross-linking ofantibodies/receptors and resulting biological effects can be avoided ifdesired.

5.1. Peptide Libraries for Use in Identifying Abtides

[0051] The abtides of the present invention can be identified from achemically synthesized peptide library or a biologically expressedpeptide library. If a biological peptide expression library is used, thenucleic acid which encodes the peptide which binds to the ligand ofchoice can be recovered, and then sequenced to determine its nucleotidesequence and hence deduce the amino acid sequence that mediates binding.Alternatively, the amino acid sequence of an appropriate binding domaincan be determined by direct determination of the amino acid sequence ofa peptide selected from a peptide library containing chemicallysynthesized peptides. In a less preferred aspect, direct amino acidsequencing of a binding peptide selected from a biological peptideexpression library can also be performed.

[0052] In a preferred embodiment of the present invention, the abtidesare advantageously identified from random peptide libraries. Typically,random peptide libraries will be encoded by synthetic oligonucleotideswith a plurality of variant nucleotide positions having the potential toencode all 20 naturally occurring amino acids. The sequence of aminoacids encoded by the variant nucleotides is unpredictable andsubstantially random. The terms “unpredicted”, “unpredictable” and“substantially random” are used interchangeably with respect to theamino acids encoded and are intended to mean that the variantnucleotides at any given position are such that it cannot be predictedwhich of the 20 naturally occurring amino acids will appear at thatposition. These variant nucleotides are the product of random chemicalsynthesis. The biological random peptide libraries envisioned for useinclude those in which a bias has been introduced into the randomsequence, e.g., to disfavor stop codon usage.

5.1.1. Chemically Synthesized Peptide Libraries

[0053] The peptide libraries used in the present invention may belibraries that are chemically synthesized in vitro. Examples of suchlibraries are given in Fodor et al., 1991, Science 251:767-773, whichdescribes the synthesis of a known array of short peptides on anindividual microscopic slide; Houghten et al., 1991, Nature 354:84-86,which describes mixtures of free hexapeptides in which the first andsecond residues in each peptide were individually and specificallydefined. Lam et al., 1991, Nature 354:82-84, which describes a splitsynthesis scheme; Medynski, 1994, Bio/Technology 12:709-710, describessplit synthesis and T-bag synthesis methods as well. See also Gallop etal., 1994, J. Medicinal Chemistry 37:1233-1251.

[0054] PCT publication WO 91/05058, dated Apr. 18, 1991, is directed torandom libraries containing semi-random nucleotide sequences. Thesemi-random nucleotide sequences are transcribed in vitro underconditions such that polysomes are produced. The polysomes are screenedfor binding to a substance of interest. Those polysomes that bind to thesubstance of interest are recovered. The RNA from those polysomes isused to construct cDNA, which is expressed to produce polypeptides.

[0055] Screening to identify peptides which bind to a ligand of choicecan be carried out by methods well known in the art.

[0056] In a specific embodiment, the total number of unpredictable aminoacids in the peptides of the chemical library used for screening isgreater than or equal to 5 and less than or equal to 25; in otherembodiments the total is in the range of 5-15 or 5-10 amino acids,preferably contiguous amino acids.

[0057] While a binding domain can be identified from chemicallysynthesized peptide libraries, such a domain would be small (i.e. lessthan 10 amino acids, and most probably. 5-6 amino acids, in length).Therefore, use of a chemically synthesized peptide library is lesspreferred for the second screening step involved in isolating abtidesthan is use in the second screening step of biological peptide librariescontaining unpredictable sequences of greater length, described below.

5.1.2. Biological Peptide Libraries

[0058] In another embodiment, biological peptide libraries are used toidentify abtides. Many suitable biological peptide libraries are knownin the art and can be used.

[0059] According to this second approach, involving recombinant DNAtechniques, peptides have been expressed in biological systems as eithersoluble fusion proteins or viral capsid fusion proteins.

[0060] A number of peptide libraries according to this approach haveused the M13 phage. Although the N-terminus of the viral capsid protein,protein III (pIII), has been shown to be necessary for viral infection,the extreme N-terminus of the mature protein does tolerate alterationssuch as insertions. Accordingly, various peptide libraries, in which thediverse peptides are expressed as pIII fusion proteins, are known in theart; these libraries can be used to identify abtides. Examples of suchlibraries are described below.

[0061] Scott and Smith, 1990, Science 249:386-390 describe constructionand expression of an “epitope library” of hexapeptides on the surface ofM13. The library was made by inserting a 33 base pair Bgl^(˜I digested oligonucleotide sequence into an Sfi I digested phage fd-tet, i.e., fUSES RF. The)33 base pair fragment contains a random or “degenerate” coding sequence(NNK)₆ where N represents G, A, T or C and K represents G or T. Theauthors stated that the library consisted of 2×10⁸ recombinantsexpressing 4×10⁷ different hekapeptides; theoretically, this libraryexpressed 69% of the 6.4×10⁷ possible peptides (20⁶). Cwirla et al.,1990, Proc. Natl. Acad. Sci. USA 87: 6378-6382 also described a somewhatsimilar library of hexapeptides expressed as pIII gene fusions of M13 fdphage. PCT publication WO 91/19818 dated Dec. 26, 1991 by Dower andCwirla describes a similar library of pentameric to octameric randomamino acid sequences.

[0062] Devlin et al. 1990, Science, 249:404-406, describes a peptidelibrary of about 15 residues generated using an (NNS) coding scheme foroligonucleotide synthesis in which S is G or C.

[0063] Christian and colleagues have described a phage display library,expressing decapeptides (Christian et al., 1992, J. Mol. Biol.227:711-718). The starting DNA was generated by means of anoligonucleotide comprising the degenerate codons [NN(G/T)]₁₀ with aself-complementary 3′ terminus. This sequence, in forming a hairpin,creates a self-priming replication site which could be used by T4 DNApolymerase to generate the complementary strand. The double-stranded DNAwas cleaved at the SfiI sites at the 5′ terminus and hairpin for cloninginto the fUSE5 vector described by Scott and Smith, supra.

[0064] Lenstra, 1992, J. Immunol. Meth. 152:149-157 describesconstruction of a library by a laborious process encompassing annealingoligonucleotides of about 17 or 23 degenerate bases with an 8 nucleotidelong palindromic sequence at their 3′ ends. This resulted in theexpression of random hexa- or octa-peptides as fusion proteins with theβ-galactosidase protein in a bacterial expression vector. The DNA wasthen converted into a double-stranded form with Klenow DNA polymerase,blunt-end ligated into a vector, and then released as Hind IIIfragments. These fragments were then cloned into an expression vector atthe sequence encoding the C-terminus of a truncated β-galactosidase togenerate 10′ recombinants.

[0065] Other biological peptide libraries which can be used includethose described in U.S. Pat. No. 5,270,170 dated Dec. 14, 1993 and PCTPublication No. WO 91/19818 dated Dec. 26, 1991. Also suitable are thosein U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,198,346, and U.S. Pat. No.5,223,409, all to Ladner et al.

[0066] The biological peptide libraries discussed above are meant to beillustrative and not limiting. It will be recognized by one of skill inthe art that many other biological peptide libraries disclosed invarious publications may be suitable for use in the practice of thepresent invention.

[0067] The protein pVIII is a major M13 viral capsid protein, which canalso serve as a site for expressing peptides on the surface of M13 viralparticles, in the construction of random peptide libraries.

[0068] While it would be understood by one skilled in the art that asfew as 5 amino acids can constitute a binding domain, the averagefunctional domain within a natural protein is considered to be about 40amino acids. Thus, the random peptide libraries from which the abtidesof the present invention are preferably identified encode peptideshaving in the range of 5 to 200 total variant amino acids. Although itis contemplated that biologically expressed random peptide librariesdisplaying short random inserts (i.e. less than 20 amino acids inlength) could be used to identify abtides of the invention, the mostpreferred biologically expressed random peptide libraries for use in theinvention are those in which the displayed peptide has 20 or greaterunpredictable amino acids i.e. preferably in the range of 20 to 100, andmost preferably 20 to 50 amino acids, as exemplified by the TSARlibraries described in PCT publication WO 91/12328, dated Aug. 22, 1991,and PCT publication WO 94/18318, dated Aug. 18, 1994.

[0069] To identify abtides, particularly in the second screening step,the invention preferably uses libraries of greater complexity than arecommonly employed in the art. The conventional teaching in the randompeptide library art is that the length of inserted oligonucleotidesshould be kept short, encoding preferably fewer than 15 and mostpreferably about 6-8 amino acids. However, not only can librariesencoding more than about 20 amino acids be constructed, but suchlibraries can be advantageously screened to identify peptides havingbinding specificity for a variety of ligands. Such libraries with longerlength inserts are exemplified by the TSAR libraries, described in PCTpublication WO 91/12328, dated Aug. 22, 1991, and PCT publication WO94/18318, dated Aug. 18, 1994.

[0070] These. PCT publications disclose that the use of librariescomposed of longer length oligonucleotides has many advantages.

[0071] Libraries composed of longer length oligonucleotides afford theability to identify peptides in which a short sequence of amino acids iscommon to or shared by a number of peptides binding a given ligand,i.e., library members having shared binding motifs. The use of longerlength libraries also affords the ability to identify peptides which donot have any shared sequences with other peptides but which neverthelesshave binding specificity for the same ligand.

[0072] When screened by the method of the present invention, librarieshaving large inserted oligonucleotide sequences provide the opportunityto identify or map binding sites which encompass not only a fewcontiguous amino acid residues, i.e., simple binding sites, but alsothose which encompass discontinuous amino acids, i.e., complex bindingsites, and may afford the complex binding characteristic of antibodiesand receptor-like molecules.

[0073] Additionally, the large size of the inserted synthesizedoligonucleotides of certain libraries provides the opportunity for thedevelopment of secondary and/or tertiary structure in the potentialbending peptides and in sequences flanking the actual binding site inthe binding domain. Secondary and tertiary structure often significantlyaffect the ability of a sequence to mediate binding, as well as thestrength and specificity of any binding which occurs. Such complexstructural effects are not possible when only small lengtholigonucleotides are used in libraries. It may be that secondary andtertiary structures are especially important in the identification ofabtides since abtides mimic the binding of large molecules such asantibodies. It is well known that the antigen binding properties ofantibodies depend in most instances upon several different regions ofthe heavy chain (complementarity determining regions) and upon regionscontributed by the light chain as well.

[0074] Therefore, it is contemplated that the most preferred bindingdomains for identifying the abtides of the present invention will bethose from biologically expressed random peptide libraries in which thedisplayed peptide is 20 or greater amino acids in length. Examples ofsuch random peptide libraries are the TSAR libraries, described in inPCT publication WO 91/12328, dated Aug. 22, 1991, and PCT publication WO94/18318, dated Aug. 18, 1994.

[0075] In one embodiment, the library utilized in the present inventionis a linear, non-constrained library. As would be understood by one inthe art having considered the present disclosure, in another specificembodiment, “constrained”, “structured” or “semi-rigid” random peptidelibraries could also be used in the present methods to identify abtides.Typically, these libraries express peptides that are substantiallyrandom but contain a small percentage of fixed residues within orflanking the random sequences that have the result of conferringstructure or some degree of conformational rigidity to the peptide. In asemirigid peptide library, the plurality of synthetic oligonucleotidesexpress peptides that are each able to adopt only one or a small numberof different conformations that are constrained by the positioning ofcodons encoding certain structure conferring amino acids in or flankingthe synthesized variant or unpredicted oligonucleotides. Unlike linear,unconstrained libraries in which the plurality of proteins expressedpotentially adopt thousands of short-lived different conformations, in asemirigid peptide library, the plurality of proteins expressed can adoptonly a single or a small number of conformations. Such libraries areexemplified by the TSAR-13 and TSAR-14 libraries described in PCTpublication WO 94/18318, dated Aug. 18, 1994; by a library of random 6amino acid sequences, each flanked by invariant cysteine residues(O'Neil et al., 1992, Proteins 14:509-515); and by those librariesdisclosed in PCT Publication No. WO 94/11496, dated May 26, 1994 (Huse).

[0076] In a preferred embodiment, a biological peptide library that is arandom peptide “TSAR” library is screened to identify an abtide. TSARsis an acronym for “Totally Synthetic Affinity Reagents” as described inPCT publication WO 91/12328, dated Aug. 22, 1991, and PCT publication WO94/18318, dated Aug. 18, 1994. TSAR libraries, their construction anduse, and specific examples of TSAR libraries, are described in detail inthose PCT publications. Nucleic acids encoding TSARs or a TSAR portionwhich mediates binding to the ligand used for screening can be used toidentify the abtides of the present invention.

[0077] A TSAR may be a heterofunctional fusion protein, said fusionprotein comprising (a) a binding domain encoded by an oligonucleotidecomprising unpredictable nucleotides in which the unpredictablenucleotides are arranged in one or more contiguous sequences, whereinthe total number of unpredictable nucleotides is greater than or equalto about 60 and less than or equal to about 600, and optionally, (b) aneffector domain encoded by an oligonucleotide sequence which is aprotein or peptide that enhances expression or detection of the bindingdomain.

[0078] Alternatively, a TSAR may be a heterofunctional fusion protein asdescribed above but in which the contiguous sequences are flanked byinvariant residues designed to encode amino acids that confer a desiredstructure to the binding domain of the expressed heterofunctional fusionprotein.

[0079] In addition to TSAR libraries, other libraries for use in thepresent invention may be those wherein the library is a library ofrecombinant vectors that express a plurality of heterofunctional fusionproteins, said fusion proteins comprising a binding domain encoded by anoligonucleotide comprising unpredictable nucleotides in which theunpredictable nucleotides are arranged in one or more contiguoussequences, wherein the total number of unpredictable nucleotides isgreater than or equal to about 15 and less than or equal to about 600.

5.2. Abtides

[0080] An abtide is typically a peptide that mimics, with respect tobinding specificity, and possibly other characteristics (e.g., bindingaffinity, sequence, etc.) a large molecule such as an antibody orreceptor. However, an abtide is generally much smaller than an antibodyor receptor. An abtide is generally a peptide of about 5 to 200 aminoacids. Preferably, an abtide is a peptide of about 10 to 100 aminoacids. Most preferably, an abtide is a peptide of about 20 to 50 aminoacids. In addition to the amino acid sequences which are responsible forthe abtide's specific binding properties, an abtide may be linked toadditional amino acid sequences or additional non-amino acid sequences.Such additional sequences may aid in the identification or isolation ofthe abtide. Or, they may aid in the biodistribution, stability, ordiagnostic or therapeutic effectiveness of the abtide when the abtide isused diagnostically or therapeutically.

[0081] The abtides may be linked to a variety of non-peptide moieties.Such moieties might include toxins; drugs; polysaccharides; nucleotides;oligonucleotides; labels such as radioactive substances (e.g. ¹¹¹In,¹²⁵I, ¹³¹I, ^(99m)Tc, ²¹²B, ⁹⁰Y, ¹⁸⁶Rh); biotin; fluorescent tags;imaging reagents (e.g. those described in U.S. Pat. No. 4,741,900 andU.S. Pat. No. 5,326,856); hydrocarbon linkers (e.g., an alkyl group orderivative thereof) conjugated to a moiety providing for attachment to asolid substratum, or to a moiety providing for easy separation (e.g., ahapten recognized by an antibody bound to a magnetic bead), etc. Linkageof the peptide to the non-peptide moiety may be by any of severalwell-known methods in the art.

[0082] In addition, in an embodiment in which an abtide has a freeamino- or carboxy-terminus, such termini can be modified by knownmethods, e.g., to provide greater resistance to degradation, greatercell permeability, greater solubility, etc., e.g., by acetylation,biotinylation, fatty acylation, etc. at the amino-terminus; amidation atthe carboxy-terminus; or the abtide can be stabilized by inclusion of Damino acids, normatural amino acids or glycosyl amino acids, etc.

[0083] The abtides of the invention are preferably made by commonlyknown methods of chemical synthesis, e.g., as described by way ofexample in Section 5.6 and its subsections.

[0084] Alternatively, abtides (or portions thereof) can be obtained byrecombinant expression of a nucleic acid encoding the desired abtide inan appropriate host, by well-known methods.

[0085] Occasionally, it may happen that not all of the amino acids inthe identified peptide will be necessary for the binding function of anabtide. Where it is desired to decrease the size of the abtide, methodscan be used to identify portions of the determined synthetic amino acidor nucleotide sequences which respectively mediate binding, or encodethe sequences which mediate binding, as described in Section 5.4 below.

5.3. Screening of Peptide Libraries to Identify Abtides

[0086] The process of identifying abtides from a peptide librarycomprises two distinct screening steps. The first step is designed toidentify epitopes or ligands with binding specificity for the largermolecule of interest, e.g. antigen mimics, receptor-ligand mimics, andthe like. In that first step, a peptide library is screened with aligand that possesses a specific, often complex, binding site ofinterest. Those peptides in the library that are specific bindingpartners of the ligand bind to the ligand and are readily recoverablebecause of this specific binding. An example of a ligand suitable foruse in this first screening step would be an antibody, inherentlypossessing an antigen binding site, or an antigen-binding derivativethereof. Another example would be a receptor e.g. the epidermal growthfactor receptor or the platelet derived growth factor receptor. Theseparticular receptors possess specific binding sites for epidermal growthfactor and platelet derived growth factor, respectively. Other receptorsare known in the art and are also potential ligands, for example, theestrogen receptor, the various acetylcholine receptors, the human growthhormone receptor, etc.

[0087] Molecules comprising those peptide sequences in the library thatare identified by the first screening step will be referred to herein asepitopes or mimetopes. For example, in the case where the first ligandis an antibody, the eptides in the library that are identified asspecific inding partners of the antibody would be known as antigenepitopes or mimetopes.

[0088] The peptides that are isolated in the first screening step arethen preferably analyzed, as, for example, by DNA sequencing of thebinding domain of the phage that encode the peptides if the library usedwas a phage library. The DNA sequence of the binding domain encodesthe-amino acid sequence of the epitope or mimetope peptide. Due to theknown relationship between DNA sequences and their encoded amino acidsequences, obtaining the DNA sequence of the epitope or mimetope allowsthe determination of the amino acid sequence of the epitope or mimetope.Alternatively, if the library used was a chemical library, direct aminoacid sequencing of the peptide epitope or mimetope can be carried out bywell known methods in the art.

[0089] In a specific embodiment, sequences of different peptidemimetopes identified in the first screening step can be compared todetermine a consensus mimetope sequence.

[0090] Once the amino acid sequence of the epitope or mimetope is known(or a portion thereof, which mediates binding), a molecule, preferably apeptide, is produced comprising that amino acid sequence which mediatesbinding. This peptide may be synthesized chemically, or, alternatively,may be produced by methods involving recombinant DNA. This peptide maycontain only the amino acids of the epitope or mimetope or, preferably,it may contain additional amino acids or non-amino acid moieties to aidin identifying or recovering the epitope or mimetope peptide and any newpeptide binders found in the subsequent screening step discussed below.

[0091] In the second screening step, the epitope or mimetope that wasidentified in the first screening step is used as a ligand for thesecond screening step. The second screening step identifies peptideswith binding specificity for the epitope or mimetope and thatsurprisingly mimic the binding specificity of the antibody or receptorthat was used as ligand in the first screening step. In other words, thesecond screening step yields peptides with antibody or receptor-likebinding activity for antigens or receptor ligands that are known asabtides.

[0092]FIG. 1 is a schematic representation of an exemplary two-stepscreening process used to identify abtides.

[0093] In a particular embodiment of the invention, it may be that theepitope to which an antibody specifically binds is known. For example,the monoclonal antibody SM-3 that specifically binds the polymorphicepithelial mucin (PEM) found on human breast carcinoma cells has beenshown to be specific for the epitope defined by the amino acid sequenceVTSAPDTRPAPGSTAPPAHGVTSAPDTR (SEQ ID NO: 9) (Burchell et al., 1989, Int.J. Cancer 44:691-696). In such cases, the first screening step describedabove may be dispensed with. A peptide comprising the sequence of theepitope for which the antibody is specific can be synthesized and usedin the second screening step described above in order to identifyabtides of the antibody.

[0094] It may also be that the portion of a “receptor-ligand” (i.e., aligand which specifically binds to a receptor) to which a receptorspecifically binds is known. For example, it has been shown thatgranulocyte/macrophage colony stimulating factor (GM-CSF) binds to theGM-CSF receptor through amino acids 88-121(HCPPTPETSCATQTITFESFKENLKDFLLVIPFDC [SEQ ID NO: 22]) of GM-CSF. Itshould be possible to synthesize a peptide Corresponding to the portionof the receptor-ligand that has been shown to be responsible forspecific binding to the receptor and to use such a peptide in the secondscreening step of the methods of the present invention in order toidentify an abtide of the receptor.

[0095] As used in the present invention, a ligand is a substance forwhich it is desired to isolate a specific binding partner from a peptidelibrary. A ligand can function as a lock, i.e., a large polypeptide orprotein analogous to a lock into which a smaller specific bindingpartner fits as a key; or a ligand can function as a key which fits intoand specifically binds a larger binding partner or lock.

[0096] In this invention, an epitope or mimetope is typically a peptidethat acts as a key; it is identified by screening a peptide library forpeptides that fit into and bind the specific binding site of a largermolecule which acts as a lock, e.g. antibody or receptor. If the largermolecule is an antibody and the peptide identified contains the portionof the amino acid sequence of the natural antigen that is responsiblefor the specific binding of the antigen to the antibody, then theidentified peptide is said to be an epitope; if the identified peptidedoes not contain the sequence of the natural antigen, then theidentified peptide is said to be a mimetope.

[0097] In a specific embodiment, a mimetope is identified by screening apeptide library with an antibody or antibody fragment. Mimetopes thusidentified functionally mimic the antigen to which the antibody binds inthat the mimetopes also specifically bind with the antibody. In somecases, if the antigen is a protein or polypeptide, the mimetopes mayshare amino acid sequence motifs with the antigen. In anotherembodiment, a mimetope is identified by screening a peptide library witha receptor or receptor fragment. Mimetopes thus identified functionallymimic the natural ligand of the receptor.

[0098] The peptide libraries that are used in the first and secondscreening steps may be the same or different. In one embodiment, apeptide library containing small random inserts (from about 6 to about15 amino acids) is used in the first screening step.

[0099] In the second screening step, it may be desirable to use a largerpeptide library. Such larger libraries preferably express peptides ofabout 20 to 200 random amino acids. Examples of such larger librariesare the TSAR libraries described in PCT publication WO 91/12328, datedAug. 22, 1991, and in PCT Publication WO 94/18318, dated Aug. 18, 1994.

[0100] Biological or chemically synthesized peptide libraries can beused in either the first or second screenings. The peptide librariesused in the present invention may have a plurality of residues that arerandom, i.e. residues for which the amino acid occupying that residuecannot be predicted in advance. Such libraries are said to be randompeptide libraries.

[0101] A preferred method for identifying abtides comprises screening alibrary of recombinant vectors that express a plurality ofheterofunctional fusion proteins, said fusion proteins comprising (a) abinding domain encoded by an oligonucleotide comprising unpredictablenucleotides in which the unpredictable nucleotides are arranged in oneor more contiguous sequences, wherein the total number of unpredictablenucleotides is greater than or equal to about 15 and less than or equalto about 600, and optionally, (b) an effector domain encoded by anoligonucleotide sequence which is a protein or peptide that enhancesexpression or detection of the binding domain. Screening is done bycontacting the plurality of heterofunctional fusion proteins with aligand under conditions conducive to ligand binding and then isolatingthe fusion proteins which bind to the ligand. The methods of theinvention further preferably comprise determining the nucleotidesequence encoding the binding domain of the heterofunctional fusionprotein identified to determine the DNA sequence that encodes thebinding domain and simultaneously to deduce the amino acid sequence ofthe mimetope used in the second screen. Nucleotide sequence analysis canbe carried out by any method known in the art, including but not limitedto the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560),the Sanger dideoxy method (Sanger et al., 1977, Proc. Natl, Acad. Sci.U.S.A. 74:5463), the use of T7 DNA polymerase (Tabor and Richardson,U.S. Pat. No. 4,795,699; Sequenase™, U.S. Biochemical Corp.), or Taqpolymerase, or use of an automated DNA sequenator (e.g., AppliedBiosystems, Foster City, Calif.).

[0102] Alternatively, the libraries used to screen for mimetopes andabtides of the invention have unpredictable nucleotides arranged in oneor more contiguous sequences that are flanked by invariant residuesdesigned to encode amino acids that confer a desired structure to thebinding domain of the expressed heterofunctional fusion protein.

[0103] Once a suitable peptide library has been constructed (orotherwise obtained), the library is screened to identify peptides havingbinding affinity for a ligand of choice. Screening the libraries can beaccomplished by any of a variety of methods known to those of skill inthe art. See, e.g., the following references, which disclose screeningof peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol.251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al.,1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl.Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt etal., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566;Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellingtonet al., 1992, Nature 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat. No.5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; and Rebarand Pabo, 1993, Science 263:671-673. See also PCT publication WO94/18318, dated Aug. 18, 1994.

[0104] If the libraries are expressed as fusion proteins with a cellsurface molecule, then screening is advantageously achieved bycontacting the vectors with an immobilized target ligand and harvestingthose vectors that bind to said ligand. Such useful screening methods,designated “panning”-techniques are described in Fowlkes et al., 1992,BioTechniques 13:422-427. In panning methods useful to screen thelibraries, the target ligand can be immobilized on plates, beads, suchas magnetic beads, sepharose, etc., or on beads used in columns. Inparticular embodiments, the immobilized target ligand can be “tagged”,e.g., using such as biotin, 2-fluorochrome, e.g. for FACS sorting.

[0105] In one embodiment, presented by way of example but notlimitation, screening a library of phage expressing random peptides onphage and phagemid vectors can be achieved by using magnetic beads asdescribed in PCT publication WO 94/18318, dated Aug. 18, 1994.

[0106] Alternatively, as yet another non-limiting example, screening alibrary of phage expressing random peptides can be achieved by panningusing microtiter plates. In a preferred method for recovering the phagebound to the wells of the microtiter plates, a pH change is used.

[0107] By way of another example, the libraries expressing randompeptides as a surface protein of either a vector or a host cell, e.g.,phage or bacterial cell, can be screened by passing a solution of thelibrary over a column of a ligand immobilized to a solid matrix, such assepharose, silica, etc., and recovering those phage that bind to thecolumn after extensive washing and elution.

[0108] By way of yet another example, weak binding library members canbe isolated based on retarded chromatographic properties. According toone mode of this embodiment for screening, fractions are collected asthey come off the column, saving the trailing fractions (i.e., thosemembers that are retarded in mobility relative to the peak fraction aresaved). These members are then concentrated and passed over the column asecond time, again saving the retarded fractions. Through successiverounds of chromatography, it is possible to isolate those that have someaffinity, albeit weak, to the immobilized ligand. These library membersare retarded in their mobility because of the millions of possibleligand interactions as the member passes down the column. In addition,this methodology selects those members that have modest affinity to thetarget, and which also have a rapid dissociation time.

[0109] If desired, the oligonucleotides encoding the binding domainselected in this manner can be mutagenized, expressed andrechromatographed (or screened by another method) to discover improvedbinding activity. In particular, saturation mutagenesis can be carriedout using synthetic oligonucleotides synthesized from “doped” nucleotidereservoirs. The doping is carried out such that the original peptidesequence is represented only once in 106 unique clones of themutagenized oligonucleotide. The assembled oligonucleotides are clonedinto a parental TSAR vector. Preferably, the vector is m663 (Fowlkes etal., 1992, BioTechniques 13:422-427). m663 is able to make blue plaqueswhen grown in E. coli stain JM101 or DH5αF′. A library of greater than10⁶ is preferred; however a library of 10⁵ is sufficient to isolate TSARphage displaying peptide domains with increased selectivity for bindingto the target ligand.

[0110] According to another alternative method, screening a library ofcan be achieved using a method comprising an “enrichment” step and afilter lift step as follows.

[0111] Random peptides from an expressed library capable of binding to agiven ligand (“positives”) are initially enriched by one or two cyclesof panning or affinity chromatography, as described above. The goal isto enrich the positives to a frequency of about >1/10⁵. Followingenrichment, a filter lift assay is conducted. For example, approximately1-2×10⁵ phage, enriched for binders, are added to 500 μl of log phase E.coli and plated on a large LB-agarose plate with 0.7w agarose in broth.The agarose is allowed to solidify, and a nitrocellulose filter (e.g.,0.45μ) is placed on the agarose surface. A series of registration marksis made with a sterile needle to allow re-alignment of the filter andplate following development as described below. Phage plaques areallowed to develop by overnight incubation at 37° C. (the presence ofthe filter does not inhibit this process). The filter is then removedfrom the plate with phage from each individual plaque adhered in situ.The filter is then exposed to a solution of BSA or other blocking agentfor 1-2 hours to prevent non-specific binding of the ligand (or“probe”).

[0112] The probe itself is labeled, for example, either by biotinylation(using commercial NHS-biotin) or direct enzyme labeling, e.g., withhorse radish peroxidase (HRP) or alkaline phosphatase. Probes labeled inthis manner are indefinitely stable and can be re-used several times.The blocked filter is exposed to a solution of probe for several hoursto allow the probe to bind in situ to any phage on the filter displayinga peptide with significant affinity to the probe. The filter is thenwashed to remove unbound probe, and then developed by exposure to enzymesubstrate solution (in the case of directly labeled probe) or furtherexposed to a solution of enzyme-labeled avidin (in the case ofbiotinylated probe). In a preferred method, an HRP-labeled probe isdetected by ECL western blotting methods (Amersham, Arlington Heights,Ill.), which involves using luminol in the presence of phenol to yieldenhanced chemiluminescence detectable by brief exposure of film byautoradiographyl in which the exposed areas of film correspond topositive plaques on the original plate. Where an enzyme substrate isused, positive phage plaques are identified by localized deposition ofcolored enzymatic cleavage product on the filter which corresponds toplaques on the original plate. The developed filter or film, as the casemay be, is simply realigned with the plate using the registration marks,and the “positive” plaques are cored from the agarose to recover thephage. Because of the high density of plaques on the original plate, itis usually impossible to isolate a single plaque from the plate on thefirst pass. Accordingly, phage recovered from the initial core arere-plated at low density and the process is repeated to allow isolationof individual plaques and hence single clones of phage.

[0113] Successful screening experiments are optimally conducted using 3rounds of serial screening. The recovered cells are then plated at a lowdensity to yield isolated colonies for individual analysis. Theindividual colonies are selected and used to inoculate LB culture mediumcontaining ampicillin. After overnight culture at 37° C., the culturesare then spun down by centrifugation. Individual cell aliquots are thenretested for binding to the target ligand attached to the beads. Bindingto other beads, having attached thereto a non-relevant ligand, can beused as a negative control.

[0114] One important aspect of screening the libraries is that ofelution. For clarity of explanation, the following is discussed in termsof TSAR expression by phage; however, it is readily understood that suchdiscussion is applicable to any system where the random peptide isexpressed on a surface fusion molecule. It is conceivable that theconditions that disrupt the peptide-target interactions during recoveryof the phage are specific for every given peptide sequence from aplurality of proteins expressed on phage. For example, certaininteractions may be disrupted by acid pH's but not by basic pH's, andvice versa. Thus, it may be desirable to test a variety of elutionconditions (including but not limited to pH 2-3, pH 12-13, excess targetin competition, detergents, mild protein denaturants, urea, varyingtemperature, light, presence or absence of metal ions, chelators, etc.)and compare the primary structures of the TSAR proteins expressed on thephage recovered for each set of conditions to determine the appropriateelution conditions for each ligand/TSAR combination. Some of theseelution conditions may be incompatible with phage infection because theyare bactericidal and will need to be removed by dialysis (i.e., dialysisbag, Centricon/Amicon microconcentrators).

[0115] The ability of different expressed proteins to be eluted underdifferent condition's may not only be due to the denaturation of thespecific peptide region involved in binding to the target but also maybe due to conformational changes in the flanking regions. These flankingsequences may also be denatured in combination with the actual bindingsequence; these flanking regions may also change their secondary ortertiary structure in response to exposure to the elution conditions(i.e., pH 2-3, pH 12-13, excess target in competition, detergents, mildprotein denaturants, urea, heat, cold, light, metal ions, chelators,etc.) which in turn leads to the conformational deformation of thepeptide responsible for binding to the target.

[0116] According to another alternative method in which the TSARscontain a linker region between the binding domain and the effectordomain, particular TSAR libraries can be prepared and screened by: (1)engineering a vector, preferably a phage vector, so that a DNA sequenceencodes a segment of Factor Xa (or Factor Xa protease cleavable peptide)and is present adjacent to the gene encoding the effector domain, e.g.,the pIII coat protein gene; (2) construct and assemble the doublestranded synthetic oligonucleotides as described above and insert intothe engineered vector; (3) express the plurality of vectors in asuitable host to form a library of vectors; (4) screen for binding to animmobilized ligand; (5) wash away excess phage; and (6) treat theimmobilized phage with Factor Xa protease. The particle will beuncoupled from the peptide-ligand complex and can then be used to infectbacteria to regenerate the particle with its full-length pIII moleculefor additional rounds of screening. This alternative embodimentadvantageously allows the use of universally effective elutionconditions and thus allows identification of phage expressing TSARs thatotherwise might not be recovered using other known methods for elution.To illustrate, using this embodiment, exceptionally tight binding TSARscould be recovered. If desired, the oligonucleotides encoding thebinding domain selected in this manner can be mutagenized, expressed andrechromatographed (or screened by another method) to discover improvedbinding activity. In particular, saturation mutagenesis can be carriedout using synthetic oligonucleotides synthesized from “doped” nucleotidereservoirs. The doping is carried out such that the original peptidesequence is represented only once in 10⁶ unique clones of themutagenized oligonucleotide. The assembled oligonucleotides are clonedinto a parental TSAR vector. Preferably, the vector is m663 (Fowlkes etal., 1992, BioTechniques 13:422-427). m663 is able to make blue plaqueswhen grown in E. coli stain JM101 or DH5αF′. A library of greater than10⁶ is preferred; however a library of 10⁵ is sufficient to isolate TSARphage displaying peptide domains with increased selectivity for bindingto the target ligand.

[0117] For the examples in Section 6 herein, a TSAR library is utilized;however, to those skilled in the art, it will be apparent that otherpeptide libraries may be used. An example of a TSAR library is theTSAR-9 library disclosed in Kay et al., 1993, Gene 128:59-65. TSAR-9constructs display a peptide of about 38 amino acids in length having 36totally random positions.

5.3.1. Antibodies and Derivatives Thereof for Use in Screening

[0118] Antibodies can be produced which recognize an antigen ofinterest. Such antibodies can be polyclonal or monoclonal. Suchantibodies may be used as ligands in the first screening step of thepresent invention.

[0119] Various procedures known in the art may be used for theproduction of polyclonal antibodies to an antigen of interest. For theproduction of antibody, various host animals can be immunized byinjection with an antigen of interest or derivative thereof, includingbut not limited to rabbits, mice, rats, etc. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, and including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet, hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and corynebacterium parvum.

[0120] A monocloinal antibody to an antigen of interest can be preparedby using any technique which provides for the production of antibodymolecules by continuous cell lines in culture. These include but are notlimited to the hybridoma technique originally described by Kohler andMilstein (1975, Nature 256:495-497), and the more recent human B cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72) andEBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0121] The monoclonal antibodies may be human monoclonal antibodies orchimeric human-mouse (or other species) monoclonal antibodies. Humanmonoclonal antibodies may be made by any of numerous techniques known inthe art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A.80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; Olsson etal., 1982, Meth. Enzymol. 92:3-16). Chimeric antibody molecules may beprepared containing a mouse antigen-binding domain with human constantregions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851,Takeda et al., 1985, Nature 314:452).

[0122] A molecular clone of an antibody to an antigen of interest can beprepared by known techniques. Recombinant DNA methodology (see e.g.,Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.) may be used toconstruct nucleic acid sequences which encode a monoclonal antibodymolecule, or antigen binding region thereof.

[0123] Antibody molecules may be purified by known techniques, e.g.,immunoabsorption or: immunoaffinity chromatography, chromatographicmethods such as HPLC (high performance liquid chromatography), or acombination thereof, etc.

[0124] Antibody fragments which contain the idiotype or antigen bindingregion of the molecule can be generated by known techniques. Forexample, such fragments include but are not limited to: the F(ab′)₂fragment which can be produced by pepsin digestion of the antibodymolecule; the Fab′ fragments which can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragment, and the 2 Fab or Fabfragments which can be generated by treating the antibody molecule withpapain and a reducing agent.

5.4. Identification of Synthetic Sequences Which Mediate Binding

[0125] When a peptide from a peptide library has been identified as anabtide or mimetope for a particular target ligand of interest accordingto the method of the invention (in either the first or second screeningstep), it may be useful to determine what region(s) of the expressedpeptide sequence is (are) responsible for binding to the target ligand.Such analysis can be conducted at two different levels, i.e., thenucleotide sequence and amino acid sequence levels.

[0126] By molecular biological techniques it is possible to verify andfurther analyze a ligand binding peptide at the level of theoligonucleotides. First, the inserted oligonucleotides can be cleavedusing appropriate restriction enzymes and religated into the originalexpression vector and the expression product of such vector screened forligand binding to identify the oligonucleotides that encode the bindingregion of the abtide or mimetope. Second, the oligonucleotides can betransferred into another vector, e.g., from phage to phagemid. The newlyexpressed fusion proteins should acquire the same binding activity ifthe domain is necessary and sufficient for binding to the ligand. Thislast approach also assesses whether or not flanking amino acid residuesencoded by the original vector influence peptide binding in any fashion.Third, the oligonucleotides can be synthesized, based on the nucleotidesequence determined for the phage in the library that encodes thebinding peptide, amplified by cloning or PCR amplification usinginternal and flanking primers, cleaved into two pieces and cloned as twohalf-binding domain fragments. In the foregoing manner, the insertedoligonucleotides are subdivided into two equal halves. If the peptidedomain important for binding is small, then one recombinant clone woulddemonstrate binding and the other would not. If neither have binding,then either both are important or the essential portion of the domainspans the middle (which can be tested by expressing just the centralregion).

[0127] Alternatively, by synthesizing peptides corresponding to thededuced sequence of the abtide or mimetope, the binding domains can beanalyzed. First, the entire peptide should be synthesized and assessedfor binding to the target ligand to verify that the peptide is necessaryand sufficient for binding. Second, short peptide fragments, forexample, overlapping 10-mers, can be synthesized, based on the aminoacid sequence of the random peptide binding domain, and tested toidentify those binding the ligand.

[0128] In addition, in certain instances, linear motifs (consensusmotifs) may become apparent after comparing the primary structures ofdifferent binding peptides from the library having binding affinity fora target ligand. The contribution of these motifs to binding can beverified with synthesized peptides in competition experiments (i.e.,determine the concentration of peptide capable of inhibiting 50% of thebinding of the phage to its target; IC₅₀). Conversely, the motif or anyregion suspected to be important for binding can be removed from ormutated within the DNA encoding the random peptide insert and thealtered displayed peptide can be retested for binding.

[0129] Furthermore, once the binding domain of a peptide has beenidentified, the binding characteristics of that peptide can be modifiedby varying the binding domain sequence to produce a related family ofpeptides with differing properties for a specific ligand.

[0130] Moreover, in a method of directed evolution, the identifiedpeptides can be improved by additional rounds of random mutagenesis,selection, and amplification of the nucleotide sequences encoding thebinding domains. Mutagenesis can be accomplished by creating and cloninga new set of oligonucleotides that differ slightly from the parentsequence, e.g., by 1-10%. Selection and amplification are achieved asdescribed above. By way of example, to verify that the isolated peptideshave improved binding characteristics, mutants and the parent phage,differing in their lacZ expression, can be processed together during thescreening experiments. Alteration of the original blue-white colorratios during the course of the screening experiment ill serve as avisual means to assess the successful selection of enhanced binders.This process can go through numerous cycles.

5.5. Uses of Abtides 5.5.1. Assays Using Abtides

[0131] The abtides of the present invention possess bindingspecificities that are similar to those of the ligands (e.g. antibodies,receptors) that are used in the first screening step of the process bywhich the abtides are identified. Consequently, the abtides may be usedin many of the same instances where the ligand of the first screeningstep might be used. For example, if the ligand used in the firstscreening step is an antibody, the abtide that is identified after thesecond screening step will bind specifically to the same antigen towhich the antibody specifically binds. Therefore, the abtide may be usedas a substitute for the antibody in many of the reactions or assays thatthe antibody could be used in. For example, the abtide could be used inimmunoassays known in the art, e.g., those designed to detect or measurethe amount of the antigen. Of course, such immunoassays may have to besuitably modified. For example, many immunoassays make use of a step inwhich a second antibody, labeled with a radioactive moiety or an enzymesuch as alkaline phosphatase, specifically binds to the first antibody.Such a second antibody would not be expected to specifically bind to theabtide. However, it would be well within the competence of one ofordinary skill in the art to fabricate another labelling moiety, perhapsa third antibody, that was able to specifically bind to the abtide, orto label the abtide with a detectable marker prior to use.

[0132] The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassays), “sandwich” immunoassays, dot immunoblot assays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, immunoaffinity chromatography, andflow dipstick assays to name but a few. For examples of exemplaryprocedures which can be used in immunoassays, see generally Kricka,1985, Clinical and Biochemical Analysis 17:1-15; Armbruster, 1993, Clin.Chem. 39/2:181-195; Birnbaum et al., 1992, Anal. Biochem. 206:168-171;Miyai, 1985, Adv. Clin. Chem. 24:61-110; and references cited therein.

[0133] The samples to be assayed in the immunoassays can be any samplethat may contain the antigen or ligand desired to be assayed. Forexample, these samples can be body fluids such as plasma, blood, serum,saliva, cerebrospinal fluid, synovial fluid, etc.

[0134] The detectable label to be used in the immunoassays can be anydetectable label known in the art. Such labels include radioisotopes,fluorescent dyes, enzymes (for example horseradish peroxidase oralkaline phosphatase), chemiluminescent molecules, metal atoms, orphosphorescent dyes, colored particles, metal and dye colloids.

5.5.1.1. Sandwich ELISA

[0135] In a particular embodiment, the abtides can be used in a sandwichenzyme immunoassay. One description of such an embodiment, presented byway of example and not limitation, follows: A molecule comprising anabtide is affixed to a solid substratum. The molecule comprising theabtide may be linked to a substance that will provide for greaterattachment of the molecule to the substratum. The sample to be assayedis contacted with the molecule comprising the abtide that is bound tothe substratum. The substances in the sample that are specific bindingpartners of the abtide (analyte) will bind to the abtide, andnon-binding sample components are removed by washing. Anenzyme-conjugated monoclonal antibody directed against the analyte isadded. This enzyme-conjugated monoclonal antibody binds to the part ofthe analyte it is specific for and completes the sandwich. After removalof unbound enzyme-conjugated monoclonal antibody by washing, a substratesolution is added to the wells. A colored product is formed inproportion to the amount of analyte present in the sample. The reactionmay be terminated by addition of stop solution and absorbance ismeasured spectrophotometrically. The following illustrates these stepsin more detail.

[0136] (a) Polystyrene microtiter wells (Flow Laboratory) are coatedovernight at room temperature with 100 μl of a solution of a moleculecomprising an abtide at a concentration of 1 mg/ml in phosphate bufferedsaline (PBS).

[0137] (b) Coating solution is discarded and wells are blocked for 1-2hours at room temperature with 300 μl of 1% bovine serum albumin (BSA)in phosphate-buffered saline (PBS) with 0.05% of Tween 20 (PBS-Tweenbuffer).

[0138] (c) 150 μl of sample (suspected of containing an analyte thepresence or amount of which it is desired to measure) diluted in 1%BSA-PBS is added per well. Wells are incubated 1 hour at roomtemperature.

[0139] (d) Wells are washed 4 times with PBS-Tween buffer.

[0140] (e) 100 μl of horseradish peroxidase conjugated monoclonalantibody specific for the analyte in 1% BSA-PBS is added per well. Theconcentration of the monoclonal antibody can be from about 10 ng/ml to10 mg/ml. Wells are incubated 1 hour at room temperature.

[0141] (f) Wells are washed 6 times with PBS-Tween buffer.

[0142] (g) 100 μl of ABTS® Boehringer Mannheim(2,2′-Azino-di-[3-ethylbenzthiazdine sulfonate (6)] crystallizeddiammonium salt working solution is added per well. ABTS® stock solutionis prepared at 15 mg/ml in dH₂O. To make the working solution, 200 μl ofthis ABTS® stock is diluted into 10 ml of citrate phosphate buffer (17mm citric acid, 65 mm dibasic sodium phosphate) and 10 μl 30% H₂O₂.

[0143] (h) The absorbance of each well is measured at 405 nm in amicrotiter plate reader (Dynatech MR600, Dynatech Corp., Alexandria,Va.).

5.5.2. Pharmaeceutical Compositions

[0144] The invention provides methods of treatment by administration toa subject of an effective amount of a pharmaceutical (therapeutic ordiagnostic) composition comprising an abtide. Such an abtide envisionedfor therapeutic or diagnostic use is referred to hereinafter as a“Therapeutic” or “Therapeutic of the invention.” Such therapeutics areabtides that specifically bind to a molecule in vivo, to exert atherapeutic or diagnostic effect. In a preferred aspect, the Therapeuticis substantially purified. The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

[0145] Formulations and methods of administration that can be employedare known in the art and can be selected from among those describedhereinbelow.

[0146] Various delivery systems are known and can be used to administera Therapeutic of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells containing theTherapeutic, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987,J. Biol. Chem. 262:4429-4432), etc. Methods of introduction include butare not limited to intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, and oral routes as wellas transdermal and subcutaneous time-release implants. The Therapeuticsmay be administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the pharmaceutical compositions of the inventioninto the central nervous system by any suitable route, includingintraventricular and intrathecal injection; intraventricular injectionmay be facilitated by an intraventricular catheter, for example,attached to a reservoir, such as an Ommaya reservoir. In a specificembodiment, it may be desirable to utilize liposomes targeted viaabtides to specific identifiable cell surface antigens.

[0147] In a specific embodiment, it may be desirable to administer theTherapeutics of the invention locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, topical application, e.g., in conjunctionwith a wound dressing after surgery, by injection, by means of acatheter, by means of a suppository, or by means of an implant, saidimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers.

[0148] The present invention provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of aTherapeutic, and a pharmaceutically acceptable carrier or excipient.Such a carrier includes but is not limited to saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Thecarrier and composition can be sterile. The formulation should suit themode of administration.

[0149] The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. The compositioncan be a liquid solution, suspension, emulsion, tablet, pill, capsule,sustained release formulation, or powder. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc.

[0150] In a preferred embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Generally, the ingredientsare supplied either separately or mixed together in unit dosage form,for example, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

[0151] The Therapeutics of the invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withfree amino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0152] The amount of the Therapeutic of the invention which will beeffective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances.

[0153] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

5.5.3. In Vivo Diagnostic and Therapeutic Uses of Abtides

[0154] Another area where abtides can be used in place of antibodies isin the imaging, detection, or treatment of disease. Current diagnosticand therapeutic methods make use of antibodies to target imaging agentsor therapeutic substances, e.g. to tumors. Since abtides possess thesame specificity of binding as antibodies, abtides can be used in placeof antibodies in such diagnostic and therapeutic methods.

[0155] Abtides may be linked to chelators such as those described inU.S. Pat. No. 4,741,900 or U.S. Pat. No. 5,326,856. The abtide-chelatorcomplex may then be radiolabeled to provide an imaging agent fordiagnosis or treatment of disease. The abtide may also be used in themethods that are disclosed in co-pending U.S. patent application Ser.No. 08/127,351 for creating a radiolabeled peptide for use in imaging orradiotherapy. This application contains a review of methods of usingpeptides in imaging agents.

[0156] In in vivo diagnostic applications, specific tissues or evenspecific cellular disorders may be imaged by administration of asufficient amount of a labeled abtide of the instant invention.

[0157] A wide variety of metal ions suitable for in vivo tissue imaginghave been tested and utilized clinically. For imaging withradioisotopes, the following characteristics are generally desirable:(a) low radiation dose to the patient; (b) high photon yield whichpermits a nuclear medicine procedure to be performed in a short timeperiod; (c) ability to be produced in sufficient quantities; (d)acceptable cost; (e) simple preparation for administration; and (f) norequirement that the patient be sequestered subsequently. Thesecharacteristics generally translate into the following: (a) theradiation exposure to the most critical organ is less than 5 rad; (b) asingle image can be obtained within-several hours after infusion; (c)the radioisotope does not decay by emission of a particle; (d) theisotope can be readily detected; and (e) the half-life is less than fourdays (Lamb and Kramer, “Commercial Production of Radioisotopes forNuclear Medicine”, In Radiotracers For Medical Applications, Vol. 1,Rayudu (Ed.), CRC Press, Inc., Boca Raton, pp. 17-62). Preferably, themetal is technetium-99m.

[0158] By way of illustration, the targets that one may image includeany solid neoplasm, certain organs such a lymph nodes, parathyroids,spleen and kidney, sites of inflammation or infection (e.g., macrophagesat such sites)., myocardial infarction or thromboses (neoantigenicdeterminants on fibrin or platelets), and the like evident to one ofordinary skill in the art. Furthermore, the neoplastic tissue may bepresent in bone, internal organs, connective tissue, or skin.

[0159] As is also apparent to one of ordinary skill in the art, one mayuse the present invention in in vivo therapeutics (e.g., usingradiotherapeutic metal complexes), especially after having diagnosed adiseased condition via the in vivo diagnostic method described above, orin in vitro diagnostic application (e.g., using a radiometal or afluorescent metal complex).

[0160] Accordingly, a method of obtaining an image of an internal regionof a subject is contemplated in the instant invention which comprisesadministering to a subject an effective amount of an abtide compositioncontaining a metal in which the metal is radioactive, and recording thescintigraphic image obtained from the decay of the radioactive metal.Likewise, a method is contemplated of enhancing an MR image of aninternal region of a subject which comprises administering to a subjectan effective amount of an abtide composition containing a metal in whichthe metal is paramagnetic, and recording the MR image of an internalregion of the subject.

[0161] Other methods include a method of enhancing a sonographic imageof an internal region of a subject comprising administering to a subjectan effective amount of an abtide composition containing a metal andrecording the sonographic image of an internal region of the subject. Inthis latter application, the metal is preferably any non-toxic heavymetal ion. A method of enhancing an X-ray image of an internal region ofa subject is also provided which comprises administering to a subject anabtide composition containing a metal, and recording the X-ray image ofan internal region of the subject. A radioactive, non-toxic heavy metalion is preferred.

[0162] The use of abtides in place of antibodies in such methods hascertain advantages. Because abtides are peptides rather than largeproteins such as antibodies, the kinetics of their distribution in thebody and clearance from the bloodstream differ from that of largeproteins such as antibodies. For example, as demonstrated in Section6.1.4, abtides can be used for in vivo imaging of disease states inabout 2 to 5 hours. Current methods of tumor imaging using antibodiesrequire approximately 24 to 48 hours.

[0163] Because abtides are peptides, they are cleared from the bloodfaster than antibodies. This means that there will be less backgroundsignal in the bloodstream when using abtides to image disease statesthan there is when using antibodies.

[0164] Peptides most likely will provoke less of an immune response inpatients than do large proteins such as antibodies. This considerationis especially important when diagnosis or treatment is required to bedone repeatedly or over a long period of time.

[0165] Abtides, because they are generally small proteins, can remainsoluble in physiological fluids under conditions where antibodiescannot.

[0166] Abtides, again because they are generally peptides, can beproduced synthetically or by recombinant methods and therefore may beless costlylto produce than antibodies.

[0167] Abtides may be used individually. Alternatively, abtides may beused as compositions of abtides in which the peptide sequences of theabtides differ.

5.6. Synthesis of Peptides 5.6.1. Procedure for Solid Phase Synthesis

[0168] Abtide or mimetope peptides may be prepared by methods that areknown in the art. For example, in brief, solid phase peptide synthesisconsists of coupling the carboxyl group of the C-terminal amino acid toa resin and successively adding N-alpha protected amino acids. Theprotecting groups may be any known in the art. Before each new aminoacid is added to the growing chain, the protecting group of the previousamino acid added to the chain is removed. The coupling of amino acids toappropriate resins is described by Rivier et al., U.S. Pat. No.4,244,946. Such solid phase syntheses have been described, for example,by Merrifield, 1964, J. Am. Chem. Soc. 85:2149; Vale et al., 1981,Science 213:1394-1397; Marki et al., 1981, J. Am. Chem. Soc. 103:3178and in U.S. Pat. Nos. 4,305,872 and 4,316,891. In a preferred aspect, anautomated peptide synthesizer is employed.

[0169] By way of example but not limitation, peptides can be synthesizedon an Applied Biosystems Inc. (“ABI”.) model 431A automated peptidesynthesizer using the “Fastmo” synthesis protocol supplied by ABI, whichuses 2-(1H-Benzotriazol-1-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphate (“HBTU”) (R. Knorr et al., 1989, Tet. Lett.,30:1927) as coupling agent. Syntheses can be carried out on 0.25 mmol ofcommercially available4-(2′,4′-dimethoxyphenyl-(9-fluorenyl-methoxycarbonyl)-aminomethyl)-phenoxypolystyrene resin (“Rink resin” from Advanced ChemTech) (H. Rink, 1987,Tet. Lett. 28:3787). Fmoc amino acids (1 mmol) are coupled according tothe Fastmoc protocol. The following side chain protected Fmoc amino acidderivatives are used: FmocArg(Pmc)OH; FmocAsn(Mbh)OH; FmocAsp(^(t)Bu)OH;FmocCys(Acm)OH; FmocGlu(tBu)OH; FmocGln(Mbh)OH; FmocHis(Tr)OH;FmocLys(Boc)OH; FmocSer(^(t)Bu)OH; FmocThr(^(t)Bu)OH; FmocTyr(^(t)Bu)OH.[Abbreviations: Acm, acetamidomethyl; Boc, tert-butoxycarbonyl; ^(t)Bu,tert-butyl; Fmoc, 9-fluorenylmethoxycarbonyl; Mbh,4,4′-dimethoxybenzhydryl; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl;Tr, trityl].

[0170] Synthesis is carried out using N-methylpyrrolidone (NMP) assolvent, with HBTU dissolved in N,N-dimethylformamide (DMF).Deprotection of the Fmoc group is effected using ca. 20% piperidine inNMP. At the end of each synthesis the amount of peptide present isassayed by ultraviolet spectroscopy. A sample of dry peptide resin (ca.3-10 mg)-is weighed, then 20% piperidine in DMA (10 mL) is added. After30 min sonication, the UV (ultraviolet) absorbance of thedibenzofulvene-piperidine adduct (formed by cleavage of the N-terminalFmoc group) is recorded at 301 nm. Peptide substitution (in mmol g⁻¹)can be calculated according to the equation:${substitution} = {\frac{A \times v}{7800 \times w} \times 1000}$

[0171] where A is the absorbance at 301 nm, v is the volume of 20%piperidine in DMA (in mL), 7800 is the extinction coefficient (inmol⁻¹dm³ cm⁻¹) of the dibenzofulvene-piperidine adduct, and w is theweight of the peptide-resin sample (in mg).

[0172] Finally, the N-terminal Fmoc group is cleaved using 20%piperidine in DMA, then acetylated using acetic anhydride and pyridinein DMA. The peptide resin is thoroughly washed with DMA, CH₂Cl₂ andfinally diethyl ether.

5.6.2. Cleavage and Deprotection

[0173] By way of example but not limitation, cleavage and deprotectioncan be carried out as follows: The air-dried peptide resin is treatedwith ethylmethyl-sulfide (EtSMe), ethanedithiol (EDT), and thioanisole(PhSMe) for approximately 20 min. prior to addition of-95% aqueoustrifluoracetic acid (TFA). A total volume of ca. 50 mL of these reagentsare used per gram of peptide-resin. The following ratio is used:TFA:EtSMe:EDT:PhSme (10:0.5:0.5:0.5). The mixture is stirred for 3 h atroom temperature under an atmosphere of N₂. The mixture is filtered andthe resin washed with TFA (2×3 mL). The combined filtrate is evaporatedin vacuo, and anhydrous diethyl ether added to the yellow/orangeresidue. The resulting white precipitate is isolated by filtration. SeeKing et al., 1990, Int. J. Peptide Protein Res. 36:255-266 regardingvarious cleavage methods.

5.6.3. Purification of the Peptides

[0174] Purification of the synthesized peptides can be carried out bystandard methods including chromatography (e.g., ion exchange, affinity,and sizing column chromatography, high performance liquid chromatography(HPLC)), centrifugation, differential solubility, or by any otherstandard technique.

5.6.4. Conjugation of Peptides to Other Molecules

[0175] The abtides of the present invention may be linked to othermolecules (e.g., a detectable label, a molecule facilitating adsorptionto a solid substratum, or a toxin, according to various embodiments ofthe invention) by methods that are well known in the art. Such methodsinclude the use of homobifunctional and heterobifunctional cross-linkingmolecules.

[0176] The homobifunctional molecules have at least two reactivefunctional groups, which are the same. The reactive functional groups ona homobifunctional molecule include, for example, aldehyde groups andactive ester groups. Homobifunctional molecules having aldehyde groupsinclude, for example, glutaraldehyde and subaraldehyde. The use ofglutaraldehyde as a cross-linking agent was disclosed by Poznansky etal., 1984, Science 223:1304-1306.

[0177] Homobifunctional molecules having at least two active ester unitsinclude esters of dicarboxylic acids and N-hydroxysuccinimide. Someexamples of such N-succinimidyl esters include disuccinimidyl suberateand dithio-bis-(succinimidyl propionate), and their soluble bis-sulfonicacid and bis-sulfonate salts such as their sodium and potassium salts.These homobifunctional reagents are available from Pierce, Rockford,Ill.

[0178] The heterobifunctional molecules have at least two differentreactive groups. Some examples of heterobifunctional reagents containingreactive disulfide bonds include N-succinimidyl3-(2-pyridyl-dithio)propionate (Carlsson et al., 1978, Biochem J.173:723-737), sodiumS-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene.N-succinimidyl 3-(2-pyridyldithio)propionate is preferred. Some examplesof heterobifunctional reagents comprising reactive groups having adouble bond that reacts with a thiol group include succinimidyl4-(N-maleimidomethyl)cyclohexahe-1-carboxylate and succinimidylm-maleimidobenzoate.

[0179] Other heterobifunctional molecules include succinimidyl3-(maleimido)propionate, sulfosuccinimidyl4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,maleimidobenzoyl-N-hydroxy-succinimide ester. The sodium sulfonate saltof succinimidyl m-maleimidobenzoate is preferred. Many of theabove-mentioned heterobifunctional reagents and their sulfonate saltsare available from Pierce.

[0180] Additional information regarding how to make and use these aswell as other polyfunctional reagents may be obtained from the followingpublications or others available in the art:

[0181] Carlsson et al., 1978, Biochem. J. 173:723-737.

[0182] Cumber et al., 1985, Methods in Enzymology 112:207-224.

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[0191] Staros, 1982, Biochem. 21:3950-3955.

[0192] Yoshitake et al., 1979, Eur. J. Biochem. 101:395-399.

[0193] Yoshitake et al., 1982, J. Biochem. 92:1413-1424.

[0194] Pilch and Czech, 1979, J. Biol. Chem. 254:3375-3381.

[0195] Novick et al., 1987, J. Biol. Chem. 262:8483-8487.

[0196] Lomant and Fairbanks, 1976, J. Mol. Biol. 104:243-261.

[0197] Hamada and Tsuruo, 1987, Anal. Biochem. 160:483-488.

[0198] Hashida et al., 1984, J. Applied Biochem. 6:56-63.

[0199] Additionally, methods of cross-linking are reviewed by Means andFeeney, 1990, Bioconjugate Chem. 1:2-12.

5.6.4.1. Biotinylation of Peptides

[0200] Methods of biotinylating peptides are well known in the art. Anyconvenient method may be employed in the practice of the invention. Forexample, the following procedure was used:

[0201] (1) dissolve 10 mg of peptide in 100 μL of 0.1% acetic acid;

[0202] (2) add 900 μL of PBS;

[0203] (3) add 3.3 mg of biotin-LC-NHS (Pierce, Rockford, Ill.);

[0204] (4) incubate for 30 minutes at room temperature;

[0205] (5) purify over a Superose 12 column (Pharmacia, Piscataway,N.J.).

6. EXAMPLES 6.1. Abtides Mimicking the Binding Specificity of MonoclonalAntibody 7E11-C5 6.1.1. Identification and Isolation of AbtidesMimicking the Binding Specificity of Monoclonal Antibody 7E11-C5

[0206] 7E11-C5 is a murine IgG1 monoclonal antibody specific for anantigen of a human prostate carcinoma, LNCaP. 7E11-C5 binds strongly tomalignant prostatic epithelium but only weakly to normal prostaticepithelium. It does not bind to non-prostatic tumors or to most normalorgans. See Horoszewicz et al., 1987, Anticancer Res. 7:927-936. 7E11-C5is also described in U.S. Pat. No. 5,162,504 issued Nov. 10, 1992.Hybridomas producing monoclonal antibody 7E11-C5 were grown as ascitesin mice and 7E11-C5 was purified from ascites fluid by Protein Aaffinity chromatography to over 90% purity as judged by sodiumdodecylsulfate polyacrylamide gel electrophoresis.

[0207] In order to identify abtides mimicking binding specificity ofmonoclonal antibody 7E11-C5, monoclonal antibody 7E11-C5 was used as thetarget ligand in a first screening of the TSAR-9 library (see Kay etal., 1993, Gene 128:59-65 and PCT publication WO 94/18318, dated Aug.18, 1994). The following-screening procedure was used. First, 7E11-CSwas bound to a well of a microtiter plate. 7E11-C5 at a concentration of11.2 mg/mL in phosphate buffered saline (PBS), pH 6.0, was diluted to100 μg per mL in 0.1×PBS pH 7.2. One hundred microliters (100 μL) ofthis dilution was added to one well of a microtiter plate, and allowedto incubate for 1-6 hours at room temperature or overnight at 40 C.After incubation, the well was washed at least 4 times with a blockingbuffer which consisted of either 1% bovine serum albumin (BSA) in PBS,1% non-fat dry milk (NFDM) in PBS, or 0.1 Tween® in either 1% BSA in PBSor 1% NFDM in PBS. Two hundred microliters of the blocking buffer wasthen added to the well and allowed to incubate for at least an hour atroom temperature.

[0208] Next, an aliquot of the TSAR-9 library was added to the wellcontaining bound 7E11-C5. An aliquot of the library containing 10¹⁰phage particles was added to the well and allowed to incubate for atleast 1 hour at room temperature. This resulted in the binding to theplate of those phage containing binding domains that bind to 7 μl-C5.After an hour, the well was washed extensively with either 1% bovineserum albumin (BSA) in PBS, 1% non-fat dry milk (NFDM) in PBS, or 0.1%Tween® in either 1% BSA in PBS or 1% NFDM in PBS.

[0209] After washing, phage bound to the 7E11-C5 antibody in the wellwere eluted by adding 100 μL of an acid solution of 0.2 M glycine-HCl,pH 2.0. After incubation from 15 minutes to 1 hour, the acid solutioncontaining eluted phage was transferred to a 1.5 mL microfuge tube, andan equal volume of 0.2 M Tris-HCl, pH 7.5 added to neutralize the acidsolution. In some cases, the neutralized phage solution was immediatelyadded to a second well containing bound 7E11-CS antibody, and thebinding and elution procedure repeated.

[0210] If it was desired that the level of enrichment be monitoredduring the-above steps, an irrelevant phage that does not bind 7E11-C5but that expresses the β-galactosidase gene was added to the aliquotfrom the TSAR-9 library. This phage gives rise to blue plaques whenplated in the presence of X-Gal and IPTG. Following a screening step,the eluted phage were plated in X-gal and IPTG. An aliquot of unscreenedphage were plated as well. The ratio of white to blue plaques wasmeasured for both phage samples. The increase in the proportion of whiteplaques (from the TSAR-9 phage that bind to 7E11-C5) to blue plaques(from the irrelevant phage) indicated the degree to which the screeningprocess enriched the population of phage for those phage that bind7E11-C5.

[0211] If it was desired that the specificity of binding be monitoredduring the above screening steps, screening was done against anirrelevant target (either BSA, mouse IgG, or plastic) in addition tobeing done against 7E11-C5. The enrichment of white plaques over blueplaques when panning was done against 7E11-C5 rather than an irrelevanttarget indicated the level of specificity of binding.

[0212] After screening, the phage were amplified by adding an aliquot ofthe eluted phage to a solution containing LB broth and competent DH5αF′E. coli cells (GIBCO BRL, Gaithersburg, Md.). Typically a 2-5 μL aliquotof the phage solution was added to 125 μL of LB broth containing a 1:50dilution of DH5αF′ E. Coli cells (about 1×10⁹ cells/ml). 3.3 mL of topagar was added to this solution, and the mixture was plated out onto aPetri dish containing agar. Often the phage were titered by makingseveral serial 1:10 dilutions, and plating out the dilution as describedabove. After incubation overnight at 37° C., the plates were evaluatedfor growth of plaques, and counted if desired. The plates were eluted byadding 3-5 mL of 100 mM NaCl, 10 mM MgCl₂, 10 mM Tris-HCl, pH 7.5 (SMbuffer) to each plate and incubating for 1-5 hours with gentle rocking.The solution was then removed from the plate, centrifuged, and eitherstored, amplified further, or analyzed.

[0213] In some cases, the phage were amplified in solution by adding 1-5μL of the phage solution to 1-5 mL of LB broth containing a 1:50 or1:100 dilution of competent DH5αF′ E. coli cells (about 1×10⁹ cells/ml.After incubation for either 6 hours or overnight, the solution wascentrifuged and the supernatant collected. In some cases, the phageparticles were precipitated with polyethylene glycol (PEG) by adding a ⅕volume of PEG to the clarified phage solution, and incubating for 1 houron ice. After centrifugation, the phage were usually reconstituted with100 μL of SM buffer.

[0214] Using the above procedures, nine different phage were isolatedthat expressed peptides containing binding domains that were capable ofbinding monoclonal antibody 7E11-C5. Molecules comprising these bindingdomains are thus mimetopes of the antigen recognized by the monoclonalantibody 7E11-C5. The binding domains of the peptides expressed by thenine phage were sequenced according to standard methods of DNAsequencing (Sequenase™, U.S. Biochemical Corp., Cleveland, Ohio). Thedetermination of those DNA sequences allowed the determination of theamino acid sequences of these mimetopes. These sequences are shown inTable 1. Examination of these amino acid sequences showed that theyshared a common motif of MYxxLH (SEQ ID NO. 10). TABLE 1SCVSHMLDTSRVYTAYANPG MYSRLH SPAVRPLTQSSA (SEQ ID No.: 11)SVQFKSISSRSMDDVVKDPGPKPA MYNRLH SKNPFTLS (SEQ ID No.: 12)YFDHTYSGPVVKNGGLVSPGVLS MYNRLH SDGGPSLAS (SEQ ID No.: 13) TVAT MHDRLHSAPGSGNLPGSYDIKPIFKAQSGALHS (SEQ ID No.: 14) T IDMPQTAST MYNMLHRNEPGGRKLSPPANDMPPALLKR (SEQ ID No.: 15) RLGNHVWREGGG MYQQLH HNFP (SEQID No.: 16) RDSAVENPSVGGEIP MYRYLH QR (SEQ ID No.: 17) PVQKEYGFGMSGASMIRLLR ETP (SEQ ID No.: 18) QKGGPGLLLYGGDS MYITLH EPG (SEQ ID No.: 19)MYxxLH (SEQ ID No.: 10) LYANPGMYSRLHSPA (SEQ ID No: 20)

[0215] In order to use the mimetopes to identify abtides, peptidescorresponding to the mimetope sequences were synthesized, and thendissolved in either water or PBS to give a final concentration of 5μg/mL. Specifically, a peptide called 7E11-9.5, with the sequenceLYANPGMYSRLHSPA (SEQ ID NO: 20) and a peptide with the sequenceGMYSRLHSPA (SEQ ID NO: 21) were synthesized.

[0216] First the mimetope peptide 7 μl-9.5 was tested for its ability tobind to the monoclonal antibody 7E11-C5. Ninety-six well plates(Immunlon 4, Dynatech, Alexandria, Va.) were coated with 50 μL of a 5μg/mL solution of the mimetope peptide 7 μl-9.5 and incubated overnightat room temperature. Following incubation, the wells were washed 4 timeswith 1% BSA in PBS. Biotinylated monoclonal antibody 7E11-C5 wasserially diluted with PBS beginning with a concentration of 29 μg/mL andvarious amounts of the monoclonal antibody were added to the wells thathad been coated with the mimetope peptide. After incubating for 1 hourat room temperature, the wells were washed four times with 1% BSA in PBSand 2 times with PBS. Then, 100 AL of a 1:2000 dilution ofExtravidin-Alkaline Phosphatase (4,250 units/mL) (Sigma, St. Louis, Mo.)in PBS was added to each well. After an hour, the plate was again washed4 times with PBS and 100 μL of a 1 mg/mL solution of the enzymesubstrate p-nitrophenyl phosphate was added to each well. Color wasallowed to develop for 15 minutes to 1 hour and absorbance was read at405 nm. FIG. 2 shows the results. It can be seen from FIG. 2 thatmonoclonal antibody 7E11-C5 binds to the synthesized mimetope 7E11-9.5in a concentration dependent manner.

[0217] Next, the mimetope peptide was used to isolate abtides from apeptide library. Fifty to one hundred microliters of the solution ofthis peptide was used to coat the wells of a 96-well plate, the wellswere blocked with 0.5% BSA in PBS, and the wells were used forscreening. An aliquot of the TSAR-9 random phage library (containingapproximately 3×10¹⁰ phage particles) was used as in the initialscreening, and 4 rounds of screening were performed. After the first tworounds, the phage were amplified. Two more rounds of screening were thenperformed. By this procedure, phage from the TSAR-9 library thatexpressed peptides capable of binding to the 7E11-9.5 mimetope peptidewere identified and isolated. The peptides containing the bindingdomains of these phage are abtides and were discovered to mimic thebinding specificities of monoclonal antibody 7E11-C5. These abtides aretermed “7E11-C5 abtides.”

[0218] Phage encoding the 7E11-C5 abtides were subjected to DNAsequencing of the nucleotide sequences encoding their binding peptidesin order to obtain the DNA and amino acid sequences of the 7E11-C5abtides. Table 2 shows the amino acid sequence of five-of the 7E11-C5abtides that had relatively high affinity for the mimetope. TABLE 2Clone Sequence 14 GIINANDPLPFWFMSPYTPGPAPIDINASRALVSNESG 17DLSRNLDFGRFLLYNAYVPGFTPTFISLTAEHLSSPKG 15CGRAYCLSGNYNIFGALFPGVSTPYADVGHDDAQSWRR 13RCSPIWGISYPFGLLSSNPGVCHSSDAETNIRNDILTT 16GHSNYCFVSTLGMPIVGFPSINARGLIHYGGSDPRLAA

[0219] The amino acid sequence shown in Table 2 for clone 14 is SEQ IDNO: 1. The amino acid sequence shown in Table 2 for clone 17 is SEQ IDNO: 2. The amino acid sequence shown in Table 2 for clone 15 is SEQ IDNO: 3. The amino acid sequence shown in Table 2 for clone 13 is SEQ IDNO: 4. The amino acid sequence shown in Table 2 for clone 16 is SEQ IDNO: 5.

[0220] It was of interest to determine whether there might be somestructural basis for the similarity in binding characteristics betweenthe monoclonal antibody 7E11-C5 and the 7E11-C5 abtides. The amino acidsequences of the complementarity determining regions (CDRs) of themonoclonal antibody 7E11-C5 were determined by sequencing cDNA clones ofthe genes encoding the variable regions of the antibody. These CDRs areresponsible for the specific binding of monoclonal antibody 7E11-C5 toits antigen. FIG. 3 presents a comparison of the amino acid sequences ofthe abtides of Table 2 and portions of the amino acid sequences of someof the CDRs of monoclonal antibody 7E11-C5. Surprisingly, it can be seenthat there are similarities in the sequences of these abtides and thesequences of the CDRs of the monoclonal antibody.

6.1.2. Characterization of 7E11′-C5 Abtides

[0221] 7E11-C5 abtides were tested for their ability to bind to the 0.7μl-C5 mimetopes that were used as target ligands in the second screeningstep above. The DNA sequences of the regions of the phage DNA encodingthe abtides were determined. This allowed the determination of the aminoacid sequences of the abtides. Based upon these determined amino acidsequences, synthetic peptides corresponding to these sequences weremade. These synthetic peptides (7E11-C5 abtides) were 38 amino acids inlength.

6.1.2.1. Dot Blots Using 7E11-C5 Abtides

[0222] In some cases, these abtides were used in a dot blot experiment.In those cases, 1 μL of a 1 mg/mL solution of the 38-residue abtides wasspotted onto nitrocellulose (0.2 μm or 0.45 μm, Schleicher & Schuell,Keene, NH) strips or circles. After drying (about {fraction (1/2)}hour), the nitrocellulose was blocked for 1 hour in a solution of 1% BSAin PBS. The nitrocellulose was then allowed to incubate in approximately5 mL of a solution of 0.1 mg/mL of a biotinylated 7E11-9.5 mimetopepeptide (biotin-LYANPGMYSRLHSPA). This mimetope peptide was one of thosedescribed in Section 6.1.1 above that were synthesized based upon thenine peptides that were identified in the screening of Section 6.1.1above. After an hour, the nitrocellulose was washed approximately 5times with a solution of 1% BSA in PBS. A 1:2000 dilution ofExtravidin-Alkaline Phosphatase (4,250 units/mL) (Sigma, St. Louis, Mo.)in PBS was then added and allowed to incubate for 1 hour, after whichthe nitrocellulose was again washed extensively. Finally, a solution of5-bromo-4-chloro-3-indolyl phosphate (0.15 mg/mL) and nitro bluetetrazolium (0.3 mg/mL) (Sigma, St Louis, Mo.) (BCIP/NBT) was added asan enzyme substrate. Color was allowed to develop and the absorbance at405 nm was read.

[0223] An example of such a dot blot assay is shown in FIG. 4. In FIG.4, the 7E11-C5 abtides known as clone 14, clone 17, clone 15, clone 16,and clone 13 were tested for their ability to bind the biotinylated7E11-9.5 mimetope peptide. Also tested, as a positive control, was themonoclonal antibody 7E11-C5. 7E11-C5 was spotted onto the region marked351 in FIG. 4. Inspection of FIG. 4 shows that at least three of theabtides (clone 14, clone. 17, and clone 15) bound the mimetope. Thisshows that these abtides are capable of mimicking the specific bindingexhibited by the monoclonal antibody 7E11-C5.

6.1.2.2. 7E11-C5 Abtides in Place of Antibodies in Immunoassays

[0224] The ability of abtides synthesized having the amino acid sequenceencoded by the random inserts of the phage that bound the 7E11-9.5mimetope was further evaluated by ELISA assay methods.

[0225] The 7E11-C5 abtides clone 14 and clone 17 (See Table 2) were eachdissolved in 0.1×PBS to give a solution of 5 μg/mL. Fifty microliters ofeach of these solutions was used to coat the wells of a 96-wellmicrotiter plate (Immulon 4, Dynatech, Alexandria, Va.) by overnightincubation at 4° C. Following this incubation, the abtide solutions wereremoved and the wells were blocked with 200 μL μl of a solution of 1%BSA in PBS. Mimetope peptides 7E11-9.5 (LYANPGMYSRLHSPA [SEQ IN NO: 20])and GMYSRLHSPA (SEQ ID NO: 21) were biotinylated as described in Section5.6.4.1 and dissolved in H₂O to give 1 mg/mL solutions. Serial 1:2dilutions were made of these solutions and these dilutions were added tothe wells of the microtiter plate containing the bound abtides. Afterincubation for 1 hour at room temperature, the wells were washed fourtimes with 1% BSA in PBS. Then a 1:2000 dilution of Extravidin-AlkalinePhosphatase (4,250 units/mL) (Sigma, St. Louis, Mo.) in PBS was added toeach well and incubated for 1 hour at room temperature. Followingincubation, the wells were washed four times with 1% BSA in PBS and thentwice in PBS. One hundred microliters of a 1 mg/mL solution ofp-nitrophenyl phosphate in diethanol amine (DEA) buffer (both fromKirkegaard & Perry Laboratories, Gaithersburg, Md.) was then added and,after incubation for 15-30 minutes at room temperature, the absorbanceof the solutions in the wells was read at 405 nm. The results are shownin FIG. 5.

[0226]FIG. 5 shows that, except for the non-linear effect at highconcentrations of mimetope, there is a good correlation between theamount of mimetope added to the wells and the absorbance at 405 nm. Theuse of antibodies in assays such as enzyme-linked immunosorbent assays(ELISAs) to measure the concentration of a substance is well known inthe art. The ability of antibodies to specifically bind their antigensis crucial to the success of such assays. Since abtides alsospecifically bind to antigens, it was of interest to determine ifabtides can be used in place of antibodies in immunoassays such asELISA-like assays to measure the concentration of a substance. Theresults of FIG. 5 show that the abtides of the present invention can beused in assays in much the same way that antibodies can be used inimmunoassays such as ELISAs.

6.1.2.3. Biotinylation of Antibodies

[0227] Methods of biotinylating antibodies are well known in the art.Any convenient method may be employed in the practice of the invention.For example, the following procedure was used:

[0228] (1) dissolve 10 mg of antibody in 1 mL of PBS;

[0229] (2) add 0.44 mg of biotin-LC-NHS (Pierce, Rockford, Ill.);

[0230] (3) incubate for 30 minutes at room temperature;

[0231] (4.) purify over a Superose 12 column (Pharmacia, Piscataway,N.J.).

6.1.3. 7E11-C5 Abtides and Monoclonal Antibody 7E11-C5 Recognize anLNSaP Antigen

[0232] The monoclonal antibody 7E11-CS recognizes a prostate specificmucin antigen of the human prostate cancer cell line LNCaP (Horoszewiczet al., 1987, Anticancer. Res. 7:927-936). To determine if 7E11-C5abtides recognize and bind the native antigen, the following experimentwas done.

[0233] A sandwich assay using the LNCaP antigen was performed. The wellsof a microtiter plate were coated with either monoclonal antibody7E11-C5, the clone 14 7E11-C5 abtide, or, as a negative control, BSA.Coating and washing was as for-the assay described in Section 6.1.2.2.One hundred microliters of a lysate of LNCaP cells was added to thewells. The LNCaP lysate was prepared as described in PCT publication WO94/18318, dated Aug. 18, 1994. Following capture of the lysate on theplate, 100 μL of a 5 μg/mL solution of biotinylated 7E11-CS monoclonalantibody was added to each well. Following incubation and washing as inSection 6.1.2.2, a 1:2000 dilution of Extravidin-Alkaline Phosphatase(4,250 units/mL) (Sigma, St. Louis, Mo.) in PBS was added to each welland incubated for 1 hour at room temperature. Following incubation, thewells were washed four times with 1% BSA in PBS and then twice in PBS.One hundred microliters of a 1 mg/mL solution of p-nitrophenyl phosphatein diethanol amine (DEA) buffer (both from Kirkegaard & PerryLaboratories, Gaithersburg, Md.) was then added and, after incubation atroom temperature for 15-30 minutes, the absorbance of the solutions inthe wells was read at 405 nm.

[0234] The results are shown in FIG. 6. FIG. 6 shows that the 7E11-C5abtide is capable of recognizing the native 7E11-C5 antigen in LNCaPlysates. This was a surprising discovery and would not have beenpredicted by those skilled in the art. It was generally felt thatscreening a library with a mimetope could yield a binder to thatmimetope. Whether such a binder could also bind the native epitope thatthe mimetope mimics was unknown. The mimetope could have represented theepitope in a loose fashion, e.g. its primary sequence could be slightlymodified; its secondary structure or factors influencing thepresentation of the mimetope could be different from those presentingthe native epitope. In such a case, the binder to the mimetope would notbe specific for the native epitope. The foregoing is presented aspossible explanation and not as a limitation of the present invention.

6.1.4. Use of 7E11-C5 Abtides in Biodistribution Studies

[0235] The 7E11 abtides described in Section 6.1 and its subsectionsabove were used in biodistribution studies to assess their ability totarget human prostate carcinoma LNCaP xenograft tumors that had beentransplanted into mice.

[0236] Male SCID mice (C.B-17/Icr Tac—SCID mice) were purchased from FoxChase (Philadelphia, Pa.) or. Taconic Farms (Germantown, N.Y.), werehoused in sterilized cages with filter bonnets, and were givenautoclaved laboratory rodent chow (Purina, St. Louis, Mo.), and filteredtap water ad libitum.

[0237] 1×10⁷ cells of the human prostate tumor line LNCaP (Horoszewiczet al., 1987, Anticancer Res. 7:927-936) were injected subcutaneously(s.c.) into the left rear flank of the mice. The cells were growing inexponential phase before harvesting and had been resuspended in 0.2 mLof sterile saline. Tumors were grown in the mice for 2-3 months beforeabtides were injected into the mice.

[0238] For biodistribution studies, abtides were modified at their aminotermini with the chelator diethylene-triamine-pentaacetic acid anhydride(DTPA-A) (Sigma, St. Louis, Mo.). Approximately 2 mg of each abtide wasinitially dissolved in an appropriate volume of 0.1% acetic acid andthen 1 mL of 0.1 M sodium bicarbonate, pH 8.0, was added. Two mg ofDTPA-A was suspended in 100 AL of dimethylsulfoxide (DMSO), and 10 μL ofthe abtide solution added to this DTPA-A suspension. After 5 minincubation at room temperature, the suspension was filtered through a0.2 μm polyvinylidene difluoride (PVDF) sample filter (Acrodisc, GelmanSciences, Inc., Ann Arbor, Mich.), and purified using a Superose-12 FPLCcolumn (Pharmacia, Piscataway, N.J.) with PBS as the running buffer.Modified peptides were stored-frozen at −20° C. or −70° C.

[0239] Abtides modified with DTPA were labeled with ¹¹¹InCl₂ as follows.0.1 to 0.5 mCi of ¹¹¹InCl₂ (Amersham, Chicago, Ill.) were firstneutralized by adding an equal volume of 0.1 M NaOAc, and then added to100 to 200 μg of the DTPA-A-modified abtide. After incubation for onehalf hour, the labeled peptide was purified using a Superose-12 FPLCcolumn (Pharmacia, Piscataway, N.J.) with PBS as the running buffer.Labeled fractions were collected in a fraction collector. Tubescontaining the labeled peptide ere pooled and used to prepare syringesfor injection into mice.

[0240] In one experiment, abtide clone 14-DPTA-¹¹¹In (see Table 2) wasinjected intravenously (i.v.) into two groups of mice bearing measurableLNCaP xenografts. About 0.2 mL of a 10 μg/mL solution of theradioactively labeled abtide in sterile saline was used. The specificactivity of the abtide was about 32 μCi/μg. Thus, the total injecteddose of radioactivity was about 120-140×10⁶ cpm.

[0241] The first group of mice was sacrificed 2 hours after injection ofthe abtide and tissues were dissected for analysis. The second group wassacrificed 4 hours after injection. Dissected tissues were weighed andthe amount of ¹¹¹In in them was determined by gamma counting. The cpmper gram of each tissue was calculated by dividing the cpm of ¹¹¹Infound in the tissue by the weight in grams of the tissue. The data arepresented as the ratio of the cpm/g in each organ to the cpm/g in blood(organ to blood ratio). This gave the ratios that are shown in Table 3and FIG. 7. TABLE 3 BIODISTRIBUTION OF ABTIDE CLONE 14-DPTA-¹¹¹In INLNCaP XENOGRAFT BEARING MICE Group 1^(a) Group 2^(b) Tissue^(c) AVGs.e.m. AVG s.e.m. Blood  1.00 0.00  1.00 0.00 Lung  0.81 0.04  1.06 0.26Spleen  0.83 0.16  1.42 0.74 Liver  0.95 0.04  2.12 0.69 Kidney-R 55.8510.22 171.73 77.97 Kidney-L 53.03 11.97 182.79 86.38 Tumor

0.80

2.85 Muscle  0.33 0.03  0.42 0.01 Testes-R  0.54 0.02  0.94 0.25Testes-L  0.68 0.24  0.88 0.24

[0242] Table 3 and FIG. 7 show that, with the exception of kidney, thehighest organ to blood ratio is found in the tumor, both at 2 hours andat 4 hours post-injection of abtide. This result shows that abtides withthe binding specificity of antibodies, e.g. that are specific for tumorantigens, can be used to localize to those tumors.

[0243] No unusual localization was seen to any non-tumor tissue or organexcept kidney. The ratio for kidney is extremely high due to the wellknown tendency of injected peptides to localize to the kidneys prior tobeing cleared from the body.

[0244] In another experiment, abtide clone 17-DPTA-¹¹¹In (see Table 2)was injected intravenously into four SCID mice bearing measurable LNCaPxenografts. Administration of xenografts was as above. About 0.2 mL of a0.1 μg/mL solution of the radioactively labeled clone 17 abtide insterile saline was injected. The specific activity of the abtide wasabout 2.4 μCi/ng. Thus, the total injected dose of radioactivity wasabout 100-110×10⁶ cpm.

[0245] In this experiment, mice were sacrificed at either 2 or 5 hourspost-injection with labeled abtide. Again, as above, the data arepresented as organ to blood ratios. As shown in Table 4 and FIG. 8,abtide clone 17-DPTA-¹¹¹In localized to LNCaP xenograft tumors in mice.TABLE 4 BIODISTRIBUTION OF ABTIDE CLONE 17-DPTA-¹¹¹In IN LNCαP-XENOGRAFTBEARING MICE GROUP 1 GROUP 2 MOUSE # MOUSE # TISSUE^(a) 1 6 2 3 BLOOD1.00 1.00 1.00 1.00 LUNG 2.52 2.57 4.33 2.47 SPLEEN 5.82 3.00 5.53 3.70LIVER-S 5.42 5.58 8.37 4.03 KIDNEY-R 235.63 234.88 321.09 106.74KIDNEY-L 563.71 220.69 424.64 104.09 TUMOR-S 10.60 15.01 8.36 2.90MUSCLE 0.93 2.96 1.16 3.04 TESTES-R 2.71 2.19 2.31 3.15 TESTES-L 1.641.14 5.36 3.41

[0246] Table 4 and FIG. 8, like Table 3 and FIG. 7, show that theinjected abtide localized to the tumor. This indicates that abtides canbe useful in the localization of tumors.

[0247] In contrast to the results of the two experiments describedabove, in which radioactively labeled abtides were shown to localize totumors, when the same experiments were done with a radioactively labeledcontrol (non-abtide) peptide (the tripeptide GYK-DPTA), no specificlocalization to tumors was observed. This can be seen in FIG. 9, whichshows the biodistribution results for experiments using the¹¹¹In-labeled control peptide.

[0248] The radiolabeled peptide conjugate GYK-DPTA-¹¹¹In was injectdintravenously into 5 SCID mice bearing measurable LNCaP xenografts. Micewere dissected 2 hours (n=2) and 5 hours (n=3) after injection of 1.5 μgof control peptide having a specific activity of 30 μCi/g. The organ toblood ratios are presented in Table 5 and FIG. 9. As shown, the controlpeptide did not selectively localize to the tumor. While the tumor toblood ratio in one mouse was 3.26, the control peptide distributedequally well to other organs (e.g. lung 3.52, spleen 3.27, liver 21.70,etc.). These results show that there was non-specific uptake of thecontrol peptide in these organs. While abtide clone 14-DPTA-¹¹¹Indemonstrated a tumor to blood ratio of only 1.85 at 2 hours (whichappears lower than that obtained with the control peptide), clone14-DPTA-¹¹¹In demonstrated specific localization to the tumor as theorgan to blood ratios in the other organs were much lower (e.g. lung0.81, spleen 0.83, liver 0.95, etc.). TABLE 5 BIODISTRIBUTION OFGYK-DPTA-¹¹¹In IN LNCaP XENOGRAFT BEARING MICE GROUP 1 GROUP 2 MOUSE #MOUSE # ORGAN/BLOOD 1 2 3 4 5 BLOOD  1.00  1.00  1.00  1.00  1.00 LUNG 2.96  3.52  0.95  2.84  2.39 SPLEEN  1.74  3.27  0.97  2.21  4.04LIVER-S  31.79  21.70  21.56  25.38  17.78 KIDNEY-R 563.87 406.27 273.82509.53 269.30 KIDNEY-L 584.69 417.98 297.65 433.97 280.50 TUMOR-S

MUSCLE  1.04  1.02  0.29  4.34  2.51 TESTES-R 603.01  58.49  0.82  2.05 2.25 TESTES-L  44.65  2.08  0.93  2.11  2.32

[0249] 6.2. Abtides Binding to a Breast Cancer Antigen

[0250] 6.2.1. Identification and Isolation of Abtides Binding ToPolymorphic Epithelial Mucin (PEM)

[0251] The monoclonal antibody SM-3 that specifically binds thepolymorphic epithelial mucin (PEM) tumor antigen found on human breastcancer cells has been shown to be specific for the epitope defined bythe amino acid sequence VTSAPDTRPAPGSTAPPAHGVTSAPDTR (SEQ ID NO: 9)(Bruchell et al., 1989, Int. J. Cancer 44:691-696). A peptide comprisingthis sequence was synthesized and used to isolate abtides from TSARpeptide libraries by methods analogous to those described above. Inthese experiments, the specific TSAR libraries used were R26, D38 andDC43. See FIGS. 10-12 for description of these libraries. Phage bound toPEM were eluted by either standard acid elution methods, stringent acidelution methods where phage were incubated with the PEM peptide for only10 minutes prior to washing and elution, or were eluted using excess PEMpeptide. Phage from each library were isolated that express peptidescapable of binding to PEM. The amino acid sequences of PEM binding phageare shown in Table 6. TABLE 6 Sequences of PEM Binding Phage Acid ElutedR26 Library A15 SFMDYFFHTPEPKPAGYPNAYTDPKHPA (SEQ ID NO: 26) A54SSSIFDYAPFSWGSAGLSNSSINVFERS (SEQ ID NO: 27) A5SASLWDALGGWTTSAVPSYPRPHQTPGR (SEQ ID NO: 28) A39SLGLPWIDVFGRSSAEPWPFGRTNLPRS (SEQ ID NO: 29) A16SVHGAFLDSFFPWAADGPHGRGRL-TSF (SEQ ID NO: 30) DC43 Library MA-8EEKQGGRWSTMMPRPWCHEGGCGFLYYDAMTKPKTPPIMRTAA (SEQ ID NO: 31) MA-21LPRPFDDASWKLRAVKESPDGCGFGSPLLFPPYSGLPTFSSCD (SEQ ID NO: 32) V22GSFESARGVTCIGNHSIGAHGCGPLRSYASFNRGSGRRH (SEQ ID NO: 33) D38 LibraryMA-32 DQIGSRPQTTSRSISGSWWENAKTLWQQDYAFSAPNAA (SEQ ID NO: 34) V23LSDAWGNFTTSYRDSAGFPSHAMTTSQGGKRNHASRFP (SEQ ID NO: 35) V21VQLDDTSPRASGQETSQSEYDARPLLSKFAIPRPWSR (SEQ ID NO: 36) V1IDSSKNRISGTGYLSFPHIRHANRRHMADDSNLAPGPS (SEQ ID NO: 37) Acid Eluted:“Stringent” DC43 Library V44 WSIGTHTGPEGKFRIPCDRSGCGGTTLTHGGLNSSPTGQHERP(SEQ ID NO: 38) V39 DPCEDGYWLSSVGRAGASIRGCGAIRRSSRTLTAEYSTRASNH (SEQ IDNO: 39) V10 GSKRSCWGTTISNYFRPVPEGCGSASSINPNTNTGRLPSLHRQ (SEQ ID NO: 40)V7 SSASSGCLGRAEHLDLDSVWGCGSQADMSRRYSPWYGRPRTGV (SEQ ID NO: 41) V4NVMWSSSKAGIRDCSQVPPGGCGPVNRHRASPPLTPFRHGSIR (SEQ ID NO: 42) D38 LibraryV45 PLTSGSSSEYRNRDDCPVYKYATNCPRLNFSPSRYSPF (SEQ ID NO: 43) V32GDAYGGIFSRPRQGLADSYIHASYTGKHFFRGPRPPTR (SEQ ID NO: 44) V27STCIGAEGEWKSFHNFLQCRDATSTSSSTLDPTALRFG (SEQ ID NO: 45) V40YSATLWDQFGSRQVELWSNRHASSAlPFASRASVLGSR (SEQ ID NO: 46) Peptide ElutedR26 Library P24 ILGWPFLTGLGDSTVHPRGRKGTDPS (SEQ ID NO: 47) P49SIPSFSMWLNQLGSAALPSKGNSQDRSD (SEQ ID NO: 48) P26SRDDIFTGGPLVLFRGSKTSNHDVHSMR (SEQ ID NO: 49) P6RAELVNWYEWFHVTAEAETPVINSHNMT (SEQ ID NO: 50) DC43 Library MP-1GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGGSRNNGP (SEQ ID NO: 51) MP-2GSASSCFPNFTARGVTVGFFGCGSPAHPAAPRVLNPATDFPAP (SEQ ID NO: 52) MP-22VFRRTARSSRPIGATVFPWYGCGNSNDETLPHHDSPPSFFLGA (SEQ ID NO: 53) MA-13NTCWTDLFWHGLPGGDLPRDGCGLPSELTTHPSRERRDASEN (SEQ ID NO: 54) D38 LibraryMP-20 IDWNWLERGQHNRGYLHSFPDAKSQPTRGPRVAPNGND (SEQ ID NO: 55) MP-30GRGSDMREHWPWSMPLILDQHANDPSPRAQSHYYSHPF (SEQ ID NO: 56)

[0252] 6.2.2. Saturation Mutagenesis of MP-1 and Identification ofAdditional PEM Binding Abtides

[0253] A saturation mutagenesis library based on one of the PEM abtides,MP-1, was constructed. Nucleotide sequences encoding the MP-1 abtidewere synthesized using a doping scheme similar to that described inSection 5.3 whereby each nucleotide was contaminated with 9% of each ofthe other 3 nucleotides (e.g. G=73% G, 9% A, 9% T, 9° C.). The resultingmutagenic oligonucleotides were used to construct a library by TSARlibrary methods described above (see FIG. 13).

[0254] The resulting library was screened to identify phage expressingabtides capable of binding to PEM. The binding of isolated phage to PEMwas confirmed by an ELISA assay. Phage that were shown to bind to PEM aswell as phage that did not bind to PEM were sequenced to determine theamino acid sequences of the expressed abtides. Table 7 shows the aminoacid sequences of these positive and negative binding phage. TABLE 7Sequence Comparison: MP-1 Binding Motif Positive Binding Sequences MP1GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGGSRNNGP (SEQ ID NO: 51) E4VSTGWSGTPRWCAPGGKQGSGCGNGPRWTTLTPDLGGTRKYGP (SEQ ID NO: 57) E7GAPLWCEKLSGTGSGGFKWPGCGSGPTYNTFTPARVGSDNKWP (SEQ ID NO: 58) E16GPPVWSAKSRWTGTGVLNWPGCGKVPSCSTYTPSRDRSRKSDP (SEQ ID NO: 59) E21GSALLTSKGCVRGPGGLMRPGCGNDRLGKSSTYAHGGWIKTGP (SEQ ID NO: 60) E33GSPVWSGDNRWRGSSPLKRPGCGNGAKCNTLKDNRKDSRKTKH (SEQ ID NO: 61) E44 GPLLPGEAAVHGARGLMRSGCGNGPTWNRLTAACRDSRNKGP (SEQ ID NO: 62) E65GSPVWMGSTRWTGHGWFRSQGCGNVPRTNSCAPAGKDSQNKGP (SEQ ID NO: 63) E73GAPVWRGNRWCSDNGELERPGCGYGPRFNILPPGRGNSRKPSP (SEQ ID NO: 64) E84GSSGWKVKHRCGGPGTLQRPGCGNLPLGHTFPPTRGGSHMEGA (SEQ ID NO: 65) E85GPRSWMGQPRGSDAGSCKWAGCGDAPMWRASTPGHGGPPNRGS (SEQ ID NO: 66) E88EALVCRGKPPWSGPAGLLWQGCGTGPVSRTFTSAQGRSRNKTS (SEQ ID NO: 67) E90GAPVVGDILWCSGARGAKWPGCGKGPTNKTFSHSRGGTQKSGL (SEQ ID NO: 68) E22GAPVSRCKPACGGFWGVNWPGCGNASMCKTFTNGHGVSSDNGH (SEQ ID NO: 69) E29GAHGYKNGSTCTGLGGWRCRGCGKGAMCNNPSPAGGAYHNQGP (SEQ ID NO: 70) E62 GPQGSEHQCCSGHWGLKFPGCGNGPICNNFTALRGASRKNGP (SEQ ID NO: 71) E64GEPVWCRHSGGRVQGGLDWLGCGDGPLRYTVTPARGGPSKHGP (SEQ ID NO: 72) E66GLSLVRGDSWGSGAGGWKRHGCGHGPMYNPQTPARGGSCTRNT (SEQ ID NO: 73) E67VSRAWSGKPRLMGSHGLNCPGCGKGHSGIMFIPDPAGSANTPP (SEQ ID NO: 74) E68CAPMWSGKPPWCVGGGVKFRGCGNRPDCNIITPRLVESRDKAL (SEQ ID NO: 75) E70ADPVCSRKPDGGGLRGLRWPGCGKGPILYNVTAARGGSRNNGP (SEQ ID NO: 76) NegativeBinding Sequences MPI GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGGSRNNGP (SEQID NO: 51) E3 GTRVPPGFALRGGRDGLSWAGCGKAPISKTYTSARGRSRKKGS (SEQ ID NO:77) E15 RSAVSEGKPREIVPGGCMWPGCGNGRKSNTLTHGPEQFQEIEP (SEQ ID NO: 78) E24SSGVGNGKPRSWAPDALNGGCGNIQFANTITPDRGGSCNQTL (SEQ ID NO: 79) E27GSSVCGGQPSGRGFGGLPGPGCGNGPTSNTLTSARGGFPNKGL (SEQ ID NO: 80) E37GAPLWQGDPADEVLGGSMIPGCGIGALSQTFTPTPGGSRKNVT (SEQ ID NO: 81) E43AGRELRQDEGEGGAGADVARLREGPICSTFTPARGGSCPSGL (SEQ ID NO: 82) E49QARVSMAISCRSGPSDLMHQGCGYGPRCNPDTTDSGGSHTNTP (SEQ ID NO: 83) E60GDPECRGKPRGRWTGSLACTGCGNGPNSKICTRARGVSRNKGP (SEQ ID NO: 84) E72STPGCSGYSGSGDPRCLTCTACGNGHTRKTLTPAHGRSTHKEP (SEQ ID NO: 85) E34GQPECRITSGCCGTDGNKWLGCGKVDMCNTLNPAVGCHGTNGS (SEQ ID NO: 86) E83REPVVGGKPWCRGPGGLRWRGCGKSQFDKIITLSRDNRRDKRP (SEQ ID NO: 87)

[0255] When the sequences shown in Table 7 are compared (seeparticularly the amino acid residues marked in boldface type), it ispossible to determine the influence of particular amino acid residues atspecific positions in the sequence on a peptide's ability to bind toPEM. Abtides that bind to PEM can be characterized by the formula:R₁R₂R₃R₄R₅R₆R₇R₈R₉R₉R₁₀R₁₁R₁₂R₁₃R₁₄R₁₅R₁₆R₁₇R₁₈R₁₉R₂₀R₂₁R₂₂R₂₃R₂₄R₂₅R₂₆R₂₇R₂₈R₂₉(SEQ ID NO: 88) R₃₀R₃₁R₃₂R₃₃R₃₄R₃₅R₃₆R₃₇R₃₈R₃₉R₄₀R₄₁R₄₂R₄₃

[0256] where:

[0257] R₁=G, C, E, or V, preferably G;

[0258] R₂=A, S, P, or L, preferably A;

[0259] R₃=P, T, H, or L, preferably P;

[0260] R₄=L, M, Q, G, A, or S;

[0261] R₅=W or Y, preferably W;

[0262] R₆=S, C, K or T, preferably S;

[0263] R₇=E, S, C, D, V, or R;

[0264] R₈=N, H, K, S, or E;

[0265] R₉=L, H, R, N, Q, T, or G;

[0266] R₁₀=W, P, R, T, or D, preferably W;

[0267] R₁₁=W, C, V, L, or G, preferably W;

[0268] R₁₂=S, T, M, or H, preferably S or T;

[0269] R₁₃=G;

[0270] R₁₄=S, A, G, N, Q, or H, preferably S;

[0271] R₁₅=W, H, G, A, or R;

[0272] R₁₆=G, T, E, P, V, or W, preferably G;

[0273] R₁₇=V, F, W, K, or A;

[0274] R₁₈=K, Q, D, E, R, or L, preferably K;

[0275] R₁₉=R, F, or S, preferably R;

[0276] R₂₀=P, S, I or H, preferably P;

[0277] R₂₁=G;

[0278] R₂₂=C;

[0279] R₂₃=G;

[0280] R₂₄=D, S, T, N, or H;

[0281] R₂₅=G, D, L, or R;

[0282] R₂₆=P or S, preferably P;

[0283] R₂₇=M, S, D, I, L, or R;

[0284] R₂₈=G, W, C, L, F, Y, or T, preferably G or W;

[0285] R₂₉=S, N, V, F, H, or R;

[0286] R₃₀=N, A, S, M, or R, preferably N;

[0287] R₃₁=F, Q, P, or V, preferably F;

[0288] R₃₂=S, V, I, K, A, or S;

[0289] R₃₃=P, A, N, or Y, preferably P;

[0290] R₃₄=G, N, or L;

[0291] R₃₅=K, R, C, Q or L, preferably K or R;

[0292] R₃₆=V, K, R, or A;

[0293] R₃₇=G, D, A, or E, preferably G;

[0294] R₃₈=S, T, P, Y or W; preferably S;

[0295] R₃₉=R, I, L, P, A or S;

[0296] R₄₀=N, K, or M, preferably N or K;

[0297] R₄₁=S, R, T, E, Q, P, Y or H;

[0298] R₄₂=G, A, S, D, N, P, Y, or K, preferably G;

[0299] R₄₃=P, H or A.

[0300] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description andaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

[0301] Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

1 103 38 amino acids amino acid single linear peptide 1 Gly Ile Ile AsnAla Asn Asp Pro Leu Pro Phe Trp Phe Met Ser Pro 1 5 10 15 Tyr Thr ProGly Pro Ala Pro Ile Asp Ile Asn Ala Ser Arg Ala Leu 20 25 30 Val Ser AsnGlu Ser Gly 35 38 amino acids amino acid single linear peptide 2 Asp LeuSer Arg Asn Leu Asp Phe Gly Arg Phe Leu Leu Tyr Asn Ala 1 5 10 15 TyrVal Pro Gly Phe Thr Pro Thr Phe Ile Ser Leu Thr Ala Glu His 20 25 30 LeuSer Ser Pro Lys Gly 35 38 amino acids amino acid single linear peptide 3Cys Gly Arg Ala Tyr Cys Leu Ser Gly Asn Tyr Asn Ile Phe Gly Ala 1 5 1015 Leu Phe Pro Gly Val Ser Thr Pro Tyr Ala Asp Val Gly His Asp Asp 20 2530 Ala Gln Ser Trp Arg Arg 35 38 amino acids amino acid single linearpeptide 4 Arg Cys Ser Pro Ile Trp Gly Ile Ser Tyr Pro Phe Gly Leu LeuSer 1 5 10 15 Ser Asn Pro Gly Val Cys His Ser Ser Asp Ala Glu Thr AsnIle Arg 20 25 30 Asn Asp Ile Leu Thr Thr 35 38 amino acids amino acidsingle linear peptide 5 Gly His Ser Asn Tyr Cys Phe Val Ser Thr Leu GlyMet Pro Ile Val 1 5 10 15 Gly Phe Pro Ser Ile Asn Ala Arg Gly Leu IleHis Tyr Gly Gly Ser 20 25 30 Asp Pro Arg Leu Ala Ala 35 9 amino acidsamino acid single linear peptide 6 Trp Gln Gly Thr His Phe Pro Tyr Thr 15 8 amino acids amino acid single linear peptide 7 Leu Val Ser Lys AsnAsp Ser Gly 1 5 8 amino acids amino acid single linear peptide 8 Gly SerAsp Asn Lys Ser Val Leu 1 5 28 amino acids amino acid single linearpeptide 9 Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr AlaPro 1 5 10 15 Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg 20 25 6amino acids amino acid single linear peptide 10 Met Tyr Xaa Xaa Leu His1 5 38 amino acids amino acid single linear peptide 11 Ser Cys Val SerHis Met Leu Asp Thr Ser Arg Val Tyr Thr Ala Tyr 1 5 10 15 Ala Asn ProGly Met Tyr Ser Arg Leu His Ser Pro Ala Val Arg Pro 20 25 30 Leu Thr GlnSer Ser Ala 35 38 amino acids amino acid single linear peptide 12 SerVal Gln Phe Lys Ser Ile Ser Ser Arg Ser Met Asp Asp Val Val 1 5 10 15Lys Asp Pro Gly Pro Lys Pro Ala Met Tyr Asn Arg Leu His Ser Lys 20 25 30Asn Pro Phe Thr Leu Ser 35 38 amino acids amino acid single linearpeptide 13 Tyr Phe Asp His Thr Tyr Ser Gly Pro Val Val Lys Asn Gly GlyLeu 1 5 10 15 Val Ser Pro Gly Val Leu Ser Met Tyr Asn Arg Leu His SerAsp Gly 20 25 30 Gly Pro Ser Leu Ala Ser 35 38 amino acids amino acidsingle linear peptide 14 Thr Val Ala Thr Met His Asp Arg Leu His Ser AlaPro Gly Ser Gly 1 5 10 15 Asn Leu Pro Gly Ser Tyr Asp Ile Lys Pro IlePhe Lys Ala Gln Ser 20 25 30 Gly Ala Leu His Ser Thr 35 38 amino acidsamino acid single linear peptide 15 Ile Asp Met Pro Gln Thr Ala Ser ThrMet Tyr Asn Met Leu His Arg 1 5 10 15 Asn Glu Pro Gly Gly Arg Lys LeuSer Pro Pro Ala Asn Asp Met Pro 20 25 30 Pro Ala Leu Leu Lys Arg 35 22amino acids amino acid single linear peptide 16 Arg Leu Gly Asn His ValTrp Arg Glu Gly Gly Gly Met Tyr Gln Gln 1 5 10 15 Leu His His Asn PhePro 20 23 amino acids amino acid single linear peptide 17 Arg Asp SerAla Val Glu Asn Pro Ser Val Gly Gly Glu Ile Pro Met 1 5 10 15 Tyr ArgTyr Leu His Gln Arg 20 23 amino acids amino acid single linear peptide18 Pro Val Gln Lys Glu Tyr Gly Phe Gly Met Ser Gly Ala Ser Met Ile 1 510 15 Arg Leu Leu Arg Glu Thr Pro 20 23 amino acids amino acid singlelinear peptide 19 Gln Lys Gly Gly Pro Gly Leu Leu Leu Tyr Gly Gly AspSer Met Tyr 1 5 10 15 Ile Thr Leu His Glu Pro Gly 20 15 amino acidsamino acid single linear peptide 20 Leu Tyr Ala Asn Pro Gly Met Tyr SerArg Leu His Ser Pro Ala 1 5 10 15 10 amino acids amino acid singlelinear peptide 21 Gly Met Tyr Ser Arg Leu His Ser Pro Ala 1 5 10 35amino acids amino acid single linear peptide 22 His Cys Pro Pro Thr ProGlu Thr Ser Cys Ala Thr Gln Thr Ile Thr 1 5 10 15 Phe Glu Ser Phe LysGlu Asn Leu Lys Asp Phe Leu Leu Val Ile Pro 20 25 30 Phe Asp Cys 35 43amino acids amino acid single linear peptide 23 Arg Glu Pro Val Val GlyGly Lys Pro Trp Cys Arg Gly Pro Gly Gly 1 5 10 15 Leu Arg Trp Arg GlyCys Gly Lys Ser Gln Phe Asp Lys Ile Ile Thr 20 25 30 Leu Ser Arg Asp AsnArg Arg Asp Lys Arg Pro 35 40 43 amino acids amino acid single linearpeptide Modified-site 1 /label= Xaa /note= “Xaa = Gly, Cys, Glu, or Val”Modified-site 2 /label= Xaa /note= “Xaa = Ala, Ser, Pro or Leu”Modified-site 3 /label= Xaa /note= “Xaa = Pro, Thr, His or Leu”Modified-site 4 /label= Xaa /note= “Xaa = Leu, Met, Gln, Gly, Ala, orSer” Modified-site 5 /label= Xaa /note= “Xaa = Trp or Tyr” Modified-site6 /label= Xaa /note= “Xaa = Ser, Cys, Lys or Thr” Modified-site 7/label= Xaa /note= “Xaa = Glu, Ser, Cys, Asp, Val, or Arg” Modified-site8 /label= Xaa /note= “Xaa = Asn, His, Lys, Ser or Glu” Modified-site 9/label= Xaa /note= “Xaa = Leu, His, Arg, Asn, Gln, Thr or Gly”Modified-site 10 /label= Xaa /note= “Xaa = Trp, Pro, Arg, Thr, or Asp”Modified-site 11 /label= Xaa /note= “Xaa = Trp, Cys, Val, Leu, or Gly”Modified-site 12 /label= Xaa /note= “Xaa = Ser, Thr, Met, or His”Modified-site 13 /label= Xaa /note= “Xaa = Gly” Modified-site 14 /label=Xaa /note= “Xaa = Ser, Ala, Gly, Asn, Gln, or His” Modified-site 15/label= Xaa /note= “Xaa = Trp, His, Gly, Ala, or Arg” Modified-site 16/label= Xaa /note= “Xaa = Gly, Thr, Glu, Pro, Val, or Trp” Modified-site17 /label= Xaa /note= “Xaa = Val, Phe, Trp, Lys, or Ala” Modified-site18 /label= Xaa /note= “Xaa = Lys, Gln, Asp, Glu, Arg, and Leu”Modified-site 19 /label= Xaa /note= “Xaa = Arg, Phe, or Ser”Modified-site 20 /label= Xaa /note= “Xaa = Pro, Ser, Ile, or His”Modified-site 21 /label= Xaa /note= “Xaa = Gly” Modified-site 22 /label=Xaa /note= “Xaa = Cys” Modified-site 23 /label= Xaa /note= “Xaa = Gly”Modified-site 24 /label= Xaa /note= “Xaa = Asp, Ser, Thr, or Asn”Modified-site 25 /label= Xaa /note= “Xaa = Gly, Asp, or Leu”Modified-site 26 /label= Xaa /note= “Xaa = Pro or Ser” Modified-site 27/label= Xaa /note= “Xaa = Met, Ser, Asp, Ile, Leu, or Arg” Modified-site28 /label= Xaa /note= “Xaa = Gly, Trp, Cys, Leu, Phe, Tyr, or Thr”Modified-site 29 /label= Xaa /note= “Xaa = Ser, Asn, Val, Phe, His orArg” Modified-site 30 /label= Xaa /note= “Xaa = Asn, Arg, Ser, Met, orArg” Modified-site 31 /label= Xaa /note= “Xaa = Phe, Gln, Pro, or Val”Modified-site 32 /label= Xaa /note= “Xaa = Ser, Val, Ile, Lys, Ala orSer” Modified-site 33 /label= Xaa /note= “Xaa = Pro, Ala, Asn, or Tyr”Modified-site 34 /label= Xaa /note= “Xaa = Gly, Asn, or Leu”Modified-site 35 /label= Xaa /note= “Xaa = Lys, Arg, Cys, Gln, or Leu”Modified-site 36 /label= Xaa /note= “Xaa = Val, Lys, Arg, or Ala”Modified-site 37 /label= Xaa /note= “Xaa = Gly, Asp, Ala, or Glu”Modified-site 38 /label= Xaa /note= “Xaa = Ser, Thr, Pro, Tyr, or Trp”Modified-site 39 /label= Xaa /note= “Xaa = Arg, Ile, Leu, Pro, Ala, orSer” Modified-site 40 /label= Xaa /note= “Xaa = Asn, Lys, or Met”Modified-site 41 /label= Xaa /note= “Xaa = Ser, Arg, Thr, Glu, Gln, Pro,Tyr, or His” Modified-site 42 /label= Xaa /note= “Xaa = Gly, Ala, Ser,Asp, Asn, Pro, Tyr, or Lys” Modified-site 43 /label= Xaa /note= “Xaa =Pro, His, or Ala” 24 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 3540 31 amino acids amino acid single linear peptide Modified-site 2/label= Xaa /note= “Xaa = Ser or Arg” Modified-site 15 /label= Xaa/note= “Xaa = Ser, Pro, Thr, or Ala” Modified-site 17 /label= Xaa /note=“Xaa = Val, Ala, Asp, Glu, or Gly” 25 Ser Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Ala 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Ser Arg 20 25 30 28 amino acids amino acidsingle linear peptide 26 Ser Phe Met Asp Tyr Phe Phe His Thr Pro Glu ProLys Pro Ala Gly 1 5 10 15 Tyr Pro Asn Ala Tyr Thr Asp Pro Lys His ProAla 20 25 28 amino acids amino acid single linear peptide 27 Ser Ser SerIle Phe Asp Tyr Ala Pro Phe Ser Trp Gly Ser Ala Gly 1 5 10 15 Leu SerAsn Ser Ser Ile Asn Val Phe Glu Arg Ser 20 25 28 amino acids amino acidsingle linear peptide 28 Ser Ala Ser Leu Trp Asp Ala Leu Gly Gly Trp ThrThr Ser Ala Val 1 5 10 15 Pro Ser Tyr Pro Arg Pro His Gln Thr Pro GlyArg 20 25 28 amino acids amino acid single linear peptide 29 Ser Leu GlyLeu Pro Trp Ile Asp Val Phe Gly Arg Ser Ser Ala Glu 1 5 10 15 Pro TrpPro Phe Gly Arg Thr Asn Leu Pro Arg Ser 20 25 27 amino acids amino acidsingle linear peptide 30 Ser Val His Gly Ala Phe Leu Asp Ser Phe Phe ProTrp Ala Ala Asp 1 5 10 15 Gly Pro His Gly Arg Gly Arg Leu Thr Ser Phe 2025 43 amino acids amino acid single linear peptide 31 Glu Glu Lys GlnGly Gly Arg Trp Ser Thr Met Met Pro Arg Pro Trp 1 5 10 15 Cys His GluGly Gly Cys Gly Phe Leu Tyr Tyr Asp Ala Met Thr Lys 20 25 30 Pro Lys ThrPro Pro Ile Met Arg Thr Ala Ala 35 40 43 amino acids amino acid singlelinear peptide 32 Leu Pro Arg Pro Phe Asp Asp Ala Ser Trp Lys Leu ArgAla Val Lys 1 5 10 15 Glu Ser Pro Asp Gly Cys Gly Phe Gly Ser Pro LeuLeu Phe Pro Pro 20 25 30 Tyr Ser Gly Leu Pro Thr Phe Ser Ser Cys Asp 3540 39 amino acids amino acid single linear peptide 33 Gly Ser Phe GluSer Ala Arg Gly Val Thr Cys Ile Gly Asn His Ser 1 5 10 15 Ile Gly AlaHis Gly Cys Gly Pro Leu Arg Ser Tyr Ala Ser Phe Asn 20 25 30 Arg Gly SerGly Arg Arg His 35 38 amino acids amino acid single linear peptide 34Asp Gln Ile Gly Ser Arg Pro Gln Thr Thr Ser Arg Ser Ile Ser Gly 1 5 1015 Ser Trp Trp Glu Asn Ala Lys Thr Leu Trp Gln Gln Asp Tyr Ala Phe 20 2530 Ser Ala Pro Asn Ala Ala 35 38 amino acids amino acid single linearpeptide 35 Leu Ser Asp Ala Trp Gly Asn Phe Thr Thr Ser Tyr Arg Asp SerAla 1 5 10 15 Gly Phe Pro Ser His Ala Met Thr Thr Ser Gln Gly Gly LysArg Asn 20 25 30 His Ala Ser Arg Phe Pro 35 37 amino acids amino acidsingle linear peptide 36 Val Gln Leu Asp Asp Thr Ser Pro Arg Ala Ser GlyGln Glu Thr Ser 1 5 10 15 Gln Ser Glu Tyr Asp Ala Arg Pro Leu Leu SerLys Phe Ala Ile Pro 20 25 30 Arg Pro Trp Ser Arg 35 38 amino acids aminoacid single linear peptide 37 Ile Asp Ser Ser Lys Asn Arg Ile Ser GlyThr Gly Tyr Leu Ser Phe 1 5 10 15 Pro His Ile Arg His Ala Asn Arg ArgHis Met Ala Asp Asp Ser Asn 20 25 30 Leu Ala Pro Gly Pro Ser 35 43 aminoacids amino acid single linear peptide 38 Trp Ser Ile Gly Thr His ThrGly Pro Glu Gly Lys Phe Arg Ile Pro 1 5 10 15 Cys Asp Arg Ser Gly CysGly Gly Thr Thr Leu Thr His Gly Gly Leu 20 25 30 Asn Ser Ser Pro Thr GlyGln His Glu Arg Pro 35 40 43 amino acids amino acid single linearpeptide 39 Asp Pro Cys Glu Asp Gly Tyr Trp Leu Ser Ser Val Gly Arg AlaGly 1 5 10 15 Ala Ser Ile Arg Gly Cys Gly Ala Ile Arg Arg Ser Ser ArgThr Leu 20 25 30 Thr Ala Glu Tyr Ser Thr Arg Ala Ser Asn His 35 40 43amino acids amino acid single linear peptide 40 Gly Ser Lys Arg Ser CysTrp Gly Thr Thr Ile Ser Asn Tyr Phe Arg 1 5 10 15 Pro Val Pro Glu GlyCys Gly Ser Ala Ser Ser Ile Asn Pro Asn Thr 20 25 30 Asn Thr Gly Arg LeuPro Ser Leu His Arg Gln 35 40 43 amino acids amino acid single linearpeptide 41 Ser Ser Ala Ser Ser Gly Cys Leu Gly Arg Ala Glu His Leu AspLeu 1 5 10 15 Asp Ser Val Trp Gly Cys Gly Ser Gln Ala Asp Met Ser ArgArg Tyr 20 25 30 Ser Pro Trp Tyr Gly Arg Pro Arg Thr Gly Val 35 40 43amino acids amino acid single linear peptide 42 Asn Val Met Trp Ser SerSer Lys Ala Gly Ile Arg Asp Cys Ser Gln 1 5 10 15 Val Pro Pro Gly GlyCys Gly Pro Val Asn Arg His Arg Ala Ser Pro 20 25 30 Pro Leu Thr Pro PheArg His Gly Ser Ile Arg 35 40 38 amino acids amino acid single linearpeptide 43 Pro Leu Thr Ser Gly Ser Ser Ser Glu Tyr Arg Asn Arg Asp AspCys 1 5 10 15 Pro Val Tyr Lys Tyr Ala Thr Asn Cys Pro Arg Leu Asn PheSer Pro 20 25 30 Ser Arg Tyr Ser Pro Phe 35 38 amino acids amino acidsingle linear peptide 44 Gly Asp Ala Tyr Gly Gly Ile Phe Ser Arg Pro ArgGln Gly Leu Ala 1 5 10 15 Asp Ser Tyr Ile His Ala Ser Tyr Thr Gly LysHis Phe Phe Arg Gly 20 25 30 Pro Arg Pro Pro Thr Arg 35 38 amino acidsamino acid single linear peptide 45 Ser Thr Cys Ile Gly Ala Glu Gly GluTrp Lys Ser Phe His Asn Phe 1 5 10 15 Leu Gln Cys Arg Asp Ala Thr SerThr Ser Ser Ser Thr Leu Asp Pro 20 25 30 Thr Ala Leu Arg Phe Gly 35 38amino acids amino acid single linear peptide 46 Tyr Ser Ala Thr Leu TrpAsp Gln Phe Gly Ser Arg Gln Val Glu Leu 1 5 10 15 Trp Ser Asn Arg HisAla Ser Ser Ala Leu Pro Phe Ala Ser Arg Ala 20 25 30 Ser Val Leu Gly SerArg 35 26 amino acids amino acid single linear peptide 47 Ile Leu GlyTrp Pro Phe Leu Thr Gly Leu Gly Asp Ser Thr Val His 1 5 10 15 Pro ArgGly Arg Lys Gly Thr Asp Pro Ser 20 25 28 amino acids amino acid singlelinear peptide 48 Ser Ile Pro Ser Phe Ser Met Trp Leu Asn Gln Leu GlySer Ala Ala 1 5 10 15 Leu Pro Ser Lys Gly Asn Ser Gln Asp Arg Ser Asp 2025 28 amino acids amino acid single linear peptide 49 Ser Arg Asp AspIle Phe Thr Gly Gly Pro Leu Val Leu Phe Arg Gly 1 5 10 15 Ser Lys ThrSer Asn His Asp Val His Ser Met Arg 20 25 28 amino acids amino acidsingle linear peptide 50 Arg Ala Glu Leu Val Asn Trp Tyr Glu Trp Phe HisVal Thr Ala Glu 1 5 10 15 Ala Glu Thr Pro Val Ile Asn Ser His Asn MetThr 20 25 43 amino acids amino acid single linear peptide 51 Gly Ala ProVal Trp Arg Gly Asn Pro Arg Trp Arg Gly Pro Gly Gly 1 5 10 15 Phe LysTrp Pro Gly Cys Gly Asn Gly Pro Met Cys Asn Thr Phe Thr 20 25 30 Pro AlaArg Gly Gly Ser Arg Asn Asn Gly Pro 35 40 43 amino acids amino acidsingle linear peptide 52 Gly Ser Ala Ser Ser Cys Phe Pro Asn Phe Thr AlaArg Gly Val Thr 1 5 10 15 Val Gly Phe Phe Gly Cys Gly Ser Pro Ala HisPro Ala Ala Pro Arg 20 25 30 Val Leu Asn Pro Ala Thr Asp Phe Pro Ala Pro35 40 43 amino acids amino acid single linear peptide 53 Val Phe Arg ArgThr Ala Arg Ser Ser Arg Pro Ile Gly Ala Thr Val 1 5 10 15 Phe Pro TrpTyr Gly Cys Gly Asn Ser Asn Asp Glu Thr Leu Pro His 20 25 30 His Asp SerPro Pro Ser Phe Phe Leu Gly Ala 35 40 42 amino acids amino acid singlelinear peptide 54 Asn Thr Cys Trp Thr Asp Leu Phe Trp His Gly Leu ProGly Gly Asp 1 5 10 15 Leu Pro Arg Asp Gly Cys Gly Leu Pro Ser Glu LeuThr Thr His Pro 20 25 30 Ser Arg Glu Arg Arg Asp Ala Ser Glu Asn 35 4038 amino acids amino acid single linear peptide 55 Ile Asp Trp Asn TrpLeu Glu Arg Gly Gln His Asn Arg Gly Tyr Leu 1 5 10 15 His Ser Phe ProAsp Ala Lys Ser Gln Pro Thr Arg Gly Pro Arg Val 20 25 30 Ala Pro Asn GlyAsn Asp 35 38 amino acids amino acid single linear peptide 56 Gly ArgGly Ser Asp Met Arg Glu His Trp Pro Trp Ser Met Pro Leu 1 5 10 15 IleLeu Asp Gln His Ala Asn Asp Pro Ser Pro Arg Ala Gln Ser His 20 25 30 TyrTyr Ser His Pro Phe 35 43 amino acids amino acid single linear peptide57 Val Ser Thr Gly Trp Ser Gly Thr Pro Arg Trp Cys Ala Pro Gly Gly 1 510 15 Lys Gln Gly Ser Gly Cys Gly Asn Gly Pro Arg Trp Thr Thr Leu Thr 2025 30 Pro Asp Leu Gly Gly Thr Arg Lys Tyr Gly Pro 35 40 43 amino acidsamino acid single linear peptide 58 Gly Ala Pro Leu Trp Cys Glu Lys LeuSer Gly Thr Gly Ser Gly Gly 1 5 10 15 Phe Lys Trp Pro Gly Cys Gly SerGly Pro Thr Tyr Asn Thr Phe Thr 20 25 30 Pro Ala Arg Val Gly Ser Asp AsnLys Trp Pro 35 40 43 amino acids amino acid single linear peptide 59 GlyPro Pro Val Trp Ser Ala Lys Ser Arg Trp Thr Gly Thr Gly Val 1 5 10 15Leu Asn Trp Pro Gly Cys Gly Lys Val Pro Ser Cys Ser Thr Tyr Thr 20 25 30Pro Ser Arg Asp Arg Ser Arg Lys Ser Asp Pro 35 40 43 amino acids aminoacid single linear peptide 60 Gly Ser Ala Leu Leu Thr Ser Lys Gly CysVal Arg Gly Pro Gly Gly 1 5 10 15 Leu Met Arg Pro Gly Cys Gly Asn AspArg Leu Gly Lys Ser Ser Thr 20 25 30 Tyr Ala His Gly Gly Trp Ile Lys ThrGly Pro 35 40 43 amino acids amino acid single linear peptide 61 Gly SerPro Val Trp Ser Gly Asp Asn Arg Trp Arg Gly Ser Ser Pro 1 5 10 15 LeuLys Arg Pro Gly Cys Gly Asn Gly Ala Lys Cys Asn Thr Leu Lys 20 25 30 AspAsn Arg Lys Asp Ser Arg Lys Thr Lys His 35 40 42 amino acids amino acidsingle linear peptide 62 Gly Pro Leu Leu Pro Gly Glu Ala Ala Val His GlyAla Arg Gly Leu 1 5 10 15 Met Arg Ser Gly Cys Gly Asn Gly Pro Thr TrpAsn Arg Leu Thr Ala 20 25 30 Ala Cys Arg Asp Ser Arg Asn Lys Gly Pro 3540 43 amino acids amino acid single linear peptide 63 Gly Ser Pro ValTrp Met Gly Ser Thr Arg Trp Thr Gly His Gly Trp 1 5 10 15 Phe Arg SerGln Gly Cys Gly Asn Val Pro Arg Thr Asn Ser Cys Ala 20 25 30 Pro Ala GlyLys Asp Ser Gln Asn Lys Gly Pro 35 40 43 amino acids amino acid singlelinear peptide 64 Gly Ala Pro Val Trp Arg Gly Asn Arg Trp Cys Ser AspAsn Gly Glu 1 5 10 15 Leu Glu Arg Pro Gly Cys Gly Tyr Gly Pro Arg PheAsn Ile Leu Pro 20 25 30 Pro Gly Arg Gly Asn Ser Arg Lys Pro Ser Pro 3540 43 amino acids amino acid single linear peptide 65 Gly Ser Ser GlyTrp Lys Val Lys His Arg Cys Gly Gly Pro Gly Thr 1 5 10 15 Leu Gln ArgPro Gly Cys Gly Asn Leu Pro Leu Gly His Thr Phe Pro 20 25 30 Pro Thr ArgGly Gly Ser His Met Glu Gly Ala 35 40 43 amino acids amino acid singlelinear peptide 66 Gly Pro Arg Ser Trp Met Gly Gln Pro Arg Gly Ser AspAla Gly Ser 1 5 10 15 Cys Lys Trp Ala Gly Cys Gly Asp Ala Pro Met TrpArg Ala Ser Thr 20 25 30 Pro Gly His Gly Gly Pro Pro Asn Arg Gly Ser 3540 43 amino acids amino acid single linear peptide 67 Glu Ala Leu ValCys Arg Gly Lys Pro Pro Trp Ser Gly Pro Ala Gly 1 5 10 15 Leu Leu TrpGln Gly Cys Gly Thr Gly Pro Val Ser Arg Thr Phe Thr 20 25 30 Ser Ala GlnGly Arg Ser Arg Asn Lys Thr Ser 35 40 43 amino acids amino acid singlelinear peptide 68 Gly Ala Pro Val Val Gly Asp Ile Leu Trp Cys Ser GlyAla Arg Gly 1 5 10 15 Ala Lys Trp Pro Gly Cys Gly Lys Gly Pro Thr AsnLys Thr Phe Ser 20 25 30 His Ser Arg Gly Gly Thr Gln Lys Ser Gly Leu 3540 43 amino acids amino acid single linear peptide 69 Gly Ala Pro ValSer Arg Cys Lys Pro Ala Cys Gly Gly Phe Trp Gly 1 5 10 15 Val Asn TrpPro Gly Cys Gly Asn Ala Ser Met Cys Lys Thr Phe Thr 20 25 30 Asn Gly HisGly Val Ser Ser Asp Asn Gly His 35 40 43 amino acids amino acid singlelinear peptide 70 Gly Ala His Gly Tyr Lys Asn Gly Ser Thr Cys Thr GlyLeu Gly Gly 1 5 10 15 Trp Arg Cys Arg Gly Cys Gly Lys Gly Ala Met CysAsn Asn Pro Ser 20 25 30 Pro Ala Gly Gly Ala Tyr His Asn Gln Gly Pro 3540 42 amino acids amino acid single linear peptide 71 Gly Pro Gln GlySer Glu His Gln Cys Cys Ser Gly His Trp Gly Leu 1 5 10 15 Lys Phe ProGly Cys Gly Asn Gly Pro Ile Cys Asn Asn Phe Thr Ala 20 25 30 Leu Arg GlyAla Ser Arg Lys Asn Gly Pro 35 40 43 amino acids amino acid singlelinear peptide 72 Gly Glu Pro Val Trp Cys Arg His Ser Gly Gly Arg ValGln Gly Gly 1 5 10 15 Leu Asp Trp Leu Gly Cys Gly Asp Gly Pro Leu ArgTyr Thr Val Thr 20 25 30 Pro Ala Arg Gly Gly Pro Ser Lys His Gly Pro 3540 43 amino acids amino acid single linear peptide 73 Gly Leu Ser LeuVal Arg Gly Asp Ser Trp Gly Ser Gly Ala Gly Gly 1 5 10 15 Trp Lys ArgHis Gly Cys Gly His Gly Pro Met Tyr Asn Pro Gln Thr 20 25 30 Pro Ala ArgGly Gly Ser Cys Thr Arg Asn Thr 35 40 43 amino acids amino acid singlelinear peptide 74 Val Ser Arg Ala Trp Ser Gly Lys Pro Arg Leu Met GlySer His Gly 1 5 10 15 Leu Asn Cys Pro Gly Cys Gly Lys Gly His Ser GlyIle Met Phe Ile 20 25 30 Pro Asp Pro Ala Gly Ser Ala Asn Thr Pro Pro 3540 43 amino acids amino acid single linear peptide 75 Cys Ala Pro MetTrp Ser Gly Lys Pro Pro Trp Cys Val Gly Gly Gly 1 5 10 15 Val Lys PheArg Gly Cys Gly Asn Arg Pro Asp Cys Asn Ile Ile Thr 20 25 30 Pro Arg LeuVal Glu Ser Arg Asp Lys Ala Leu 35 40 43 amino acids amino acid singlelinear peptide 76 Ala Asp Pro Val Cys Ser Arg Lys Pro Asp Gly Gly GlyLeu Arg Gly 1 5 10 15 Leu Arg Trp Pro Gly Cys Gly Lys Gly Pro Ile LeuTyr Asn Val Thr 20 25 30 Ala Ala Arg Gly Gly Ser Arg Asn Asn Gly Pro 3540 43 amino acids amino acid single linear peptide 77 Gly Thr Arg ValPro Pro Gly Phe Ala Leu Arg Gly Gly Arg Asp Gly 1 5 10 15 Leu Ser TrpAla Gly Cys Gly Lys Ala Pro Ile Ser Lys Thr Tyr Thr 20 25 30 Ser Ala ArgGly Arg Ser Arg Lys Lys Gly Ser 35 40 43 amino acids amino acid singlelinear peptide 78 Arg Ser Ala Val Ser Glu Gly Lys Pro Arg Glu Ile ValPro Gly Gly 1 5 10 15 Cys Met Trp Pro Gly Cys Gly Asn Gly Arg Lys SerAsn Thr Leu Thr 20 25 30 His Gly Pro Glu Gln Phe Gln Glu Ile Glu Pro 3540 42 amino acids amino acid single linear peptide 79 Ser Ser Gly ValGly Asn Gly Lys Pro Arg Ser Trp Ala Pro Asp Ala 1 5 10 15 Leu Asn GlyGly Cys Gly Asn Ile Gln Phe Ala Asn Thr Ile Thr Pro 20 25 30 Asp Arg GlyGly Ser Cys Asn Gln Thr Leu 35 40 43 amino acids amino acid singlelinear peptide 80 Gly Ser Ser Val Cys Gly Gly Gln Pro Ser Gly Arg GlyPhe Gly Gly 1 5 10 15 Leu Pro Gly Pro Gly Cys Gly Asn Gly Pro Thr SerAsn Thr Leu Thr 20 25 30 Ser Ala Arg Gly Gly Phe Pro Asn Lys Gly Leu 3540 43 amino acids amino acid single linear peptide 81 Gly Ala Pro LeuTrp Gln Gly Asp Pro Ala Asp Glu Val Leu Gly Gly 1 5 10 15 Ser Met IlePro Gly Cys Gly Ile Gly Ala Leu Ser Gln Thr Phe Thr 20 25 30 Pro Thr ProGly Gly Ser Arg Lys Asn Val Thr 35 40 42 amino acids amino acid singlelinear peptide 82 Ala Gly Arg Glu Leu Arg Gln Asp Glu Gly Glu Gly GlyAla Gly Ala 1 5 10 15 Asp Val Ala Arg Leu Arg Glu Gly Pro Ile Cys SerThr Phe Thr Pro 20 25 30 Ala Arg Gly Gly Ser Cys Pro Ser Gly Leu 35 4043 amino acids amino acid single linear peptide 83 Gln Ala Arg Val SerMet Ala Ile Ser Cys Arg Ser Gly Pro Ser Asp 1 5 10 15 Leu Met His GlnGly Cys Gly Tyr Gly Pro Arg Cys Asn Pro Asp Thr 20 25 30 Thr Asp Ser GlyGly Ser His Thr Asn Thr Pro 35 40 43 amino acids amino acid singlelinear peptide 84 Gly Asp Pro Glu Cys Arg Gly Lys Pro Arg Gly Arg TrpThr Gly Ser 1 5 10 15 Leu Ala Cys Thr Gly Cys Gly Asn Gly Pro Asn SerLys Ile Cys Thr 20 25 30 Arg Ala Arg Gly Val Ser Arg Asn Lys Gly Pro 3540 43 amino acids amino acid single linear peptide 85 Ser Thr Pro GlyCys Ser Gly Tyr Ser Gly Ser Gly Asp Pro Arg Cys 1 5 10 15 Leu Thr CysThr Ala Cys Gly Asn Gly His Thr Arg Lys Thr Leu Thr 20 25 30 Pro Ala HisGly Arg Ser Thr His Lys Glu Pro 35 40 43 amino acids amino acid singlelinear peptide 86 Gly Gln Pro Glu Cys Arg Ile Thr Ser Gly Cys Cys GlyThr Asp Gly 1 5 10 15 Asn Lys Trp Leu Gly Cys Gly Lys Val Asp Met CysAsn Thr Leu Asn 20 25 30 Pro Ala Val Gly Cys His Gly Thr Asn Gly Ser 3540 56 base pairs nucleic acid single linear DNA 87 CTGTGCCTCG AGBNNBNNBNNBNNBNNBNN BNNBNNBNNB NNBNNBNNBN CCGCGG 56 56 base pairs nucleic acidsingle linear DNA 88 CTGTGCTCTA GAVNNVNNVN NVNNVNNVNN VNNVNNVNNVNNVNNVNNVN CCGCGG 56 49 base pairs nucleic acid single linear DNA 89TCGAGBNNBN NBNNBNNBNN BNNBNNBNNB NNBNNBNNBN NBNCCGCGG 49 49 base pairsnucleic acid single linear DNA 90 CTAGTVNNVN NVNNVNNVNN VNNVNNVNNVNNVNNVNNVN NVNCCGCGG 49 38 amino acids amino acid single linear peptideModified-site 5 /label= Xaa /note= “Xaa = Ser or Arg” Modified-site 18/label= Xaa /note= “Xaa = Ser, Pro, Thr, or Ala” Modified-site 20/label= Xaa /note= “Xaa = Val, Ala, Asp, Glu, or Gly” 91 Ser His Ser SerXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa AlaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Ser Arg ProSer Arg Thr 35 81 base pairs nucleic acid single linear DNA 92GTGTGTCTCG AGNNNBNNBN NBNNBNNBNN BNNBNNBNNB NNBNNBNNBN NBNNBNNBNN 60BNNBNNBNNB NNBNACGCCA N 81 66 base pairs nucleic acid single linear DNA93 GTTGTGTCTA GAVNNVNNVN NVNNVNNVNN VNNVNNVNNV NNVNNVNNVN NVNNVNNVNT 60GGCGTN 66 74 base pairs nucleic acid single linear DNA 94 TCGAGNNNBNNBNNBNNBNN BNNBNNBNNB NNBNNBNNBN NBNNBNNBNN BNNBNNBNNB 60 NNBNNBNACGCCAN 74 59 base pairs nucleic acid single linear DNA 95 CTAGAVNNVNNVNNVNNVNN VNNVNNVNNV NNVNNVNNVN NVNNVNNVNN VNTGGCGTN 59 44 amino acidsamino acid single linear peptide Modified-site 4 /label= Xaa /note= “Xaa= Ser or Arg” Modified-site 25 /label= Xaa /note= “Xaa = Tyr, His, Asnor Asp” Modified-site 27 /label= Xaa /note= “Xaa = Ile, Met, Thr, Asn,Lys, Ser, or Arg” 96 His Ser Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa XaaXaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Arg35 40 82 base pairs nucleic acid single linear DNA 97 GTGTGTCTCGAGNNNBNNBN NBNNBNNBNN BNNBNNBNNB NNBNNBNNBN NBNNBNNBNN 60 BNNBNNBNNBNNBGGTTGTG GT 82 81 base pairs nucleic acid single linear DNA 98GTTGTGTCTA GAVNNVNNVN NVNNVNNVNN VNNVNNVNNV NNVNNVNNVN NVNNVNNVNN 60VNNVNNVNNV NNACCACAAC C 81 75 base pairs nucleic acid single linear DNA99 TCGAGNNNBN NBNNBNNBNN BNNBNNBNNB NNBNNBNNBN NBNNBNNBNN BNNBNNBNNB 60NNBNNBGGTT GTGGT 75 74 base pairs nucleic acid single linear DNA 100CTAGAVNNVN NVNNVNNVNN VNNVNNVNNV NNVNNVNNVN NVNNVNNVNN VNNVNNVNNV 60NNVNNACCAC AACC 74 49 amino acids amino acid single linear peptideModified-site 4 /label= Xaa /note= “Xaa = Ser or Arg” 101 His Ser SerXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Gly Cys Gly Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser 35 40 45 Arg 69base pairs nucleic acid single linear DNA 102 GGSGCSCCSG TSTGSAGSGGSAASCCSCGS TGSAGSGGSC CSGGSGGSTT SAASTGSCCS 60 GGCTGCGGG 69 69 basepairs nucleic acid single linear DNA 103 SGGSCCSTTS TTSCGSGASCCSCCSCGSGC SGGSGTSAAS GTSTTSCASA TSGGSCCSTT 60 CCCGCAGCC 69

What is claimed is:
 1. A molecule comprising a peptide which binds to asubstance of interest, which peptide is identified by a methodcomprising: (a) screening a first random peptide library with a firstligand, said first ligand being a specific binding partner of saidsubstance of interest, to identify a first peptide that specificallybinds to said first ligand; and (b) screening a second random peptidelibrary with a second ligand comprising said first peptide identified instep (a), to identify a second peptide which binds to said second ligandand which binds to said substance of interest.
 2. A molecule comprisinga peptide which binds to an antigen of interest, which peptide isidentified by a method comprising: (a) screening a first random peptidelibrary with an antibody or antigen-binding derivative thereof thatspecifically binds to an antigen of interest, to identify a firstpeptide that specifically binds to said antibody or antigen-bindingderivative thereof; and (b) screening a second random peptide librarywith a compound comprising said first peptide identified in step (a), toidentify a second peptide which binds to said compound and which bindsto said antigen of interest.
 3. The molecule of claim 2, in which saidfirst random peptide library is a different library from said secondrandom peptide library.
 4. The molecule of claim 2, in which said firstrandom peptide library is the same library as said second random peptidelibrary.
 5. The molecule of claim 1, in which said method furthercomprises comparing the sequences of a plurality of different firstpeptides identified as binding said first ligand in step (a.), toidentify a consensus binding sequence, in which said second ligand ofstep (b) comprises said consensus binding sequence.
 6. The molecule ofclaim 2, in which said method further comprises comparing the sequencesof a plurality of different first peptides identified as binding saidantibody or antigen-binding derivative thereof in step (a), to identifya consensus binding sequence, in which said compound of step (b)comprises said consensus binding sequence.
 7. The molecule of claim 1 inwhich the first ligand comprises a receptor.
 8. The molecule of claim 2in which the antibody is the monoclonal antibody 7E11-C5.
 9. Themolecule of claim 1 in which the library of step (a) or step (b) is alibrary of recombinant vectors that express a plurality ofheterofunctional fusion proteins, said fusion proteins comprising abinding domain encoded by an oligonucleotide comprising unpredictablenucleotides in which the unpredictable nucleotides are arranged in oneor more contiguous sequences, wherein the total number of unpredictablenucleotides is greater than or equal to about 15 and less than or equalto about
 600. 10. The molecule of claim 2 in which the library of step(a) or step (b) is a library of recombinant vectors that express aplurality of heterofunctional fusion proteins, said fusion proteinscomprising a binding domain encoded by an oligonucleotide comprisingunpredictable nucleotides in which the unpredictable nucleotides arearranged in one or more contiguous sequences, wherein the total numberof unpredictable nucleotides is greater than or equal to about 15 andless than or equal to about
 600. 11. The molecule of claim 1 in whichthe library of step (a) or step (b) is a chemically synthesized library.12. A molecule comprising: an amino acid sequence 10 selected from thegroup consisting of: (SEQ ID NO: 1)GIINANDPLPFWFMSPYTPGPAPIDINASRALVSNESG, (SEQ ID NO: 2)CGRAYCLSGNYNIFGALFPGVSTPYADVGHDDAQSWRR, (SEQ ID NO: 3)DLSRNLDFGRFLLYNAYVPGFTPTFISLTAEHLSSPKG, (SEQ ID NO: 4)RCSPIWGISYPFGLLSSNPGVCHSSDAETNIRNDILTT, and (SEQ ID NO: 5)GHSNYCFVSTLGMPIVGFPSINARGLIHYGGSDPRLAA;

or a binding portion thereof.
 13. A peptide in-which the amino acidsequence of said peptide consists of the sequence selected from thegroup consisting of: (SEQ ID NO: 1)GIINANDPLPFWFMSPYTPGPAPIDINASRALVSNESG, (SEQ ID NO: 2)CGPAYCLSGNYNIFGALFPGVSTPYADVGHDDAQSWRR, (SEQ ID NO: 3)DLSRNLDFGRFLLYNAYVPGFTPTFISLTAEHLSSPKG, (SEQ ID NO: 4)RCSPIWGISYPFGLLSSNPGVCHSSDAETNIRNDILTT, and (SEQ ID NO: 5)GHSNYCFVSTLGMPIVGFPSINARGLIHYGGSDPRLAA;

or a binding portion thereof.
 14. A method of identifying a peptidewhich binds to a substance of interest, comprising: (a) screening afirst random peptide library with a ligand, said ligand being a specificbinding partner of said substance of interest, to identify a firstpeptide that specifically binds to said ligand; and (b) screening asecond random peptide library with a compound comprising said firstpeptide identified in step (a), to identify a second peptide which bindsto said compound and which binds to said substance of interest.
 15. Amethod of identifying a peptide which binds to an antigen of interestcomprising: (a) screening a first random peptide library with anantibody or antigen-binding derivative thereof that specifically bindsto an antigen of interest, to identify a first peptide that specificallybinds to said antibody or antigen-binding derivative thereof; and (b)screening a second random peptide library with a molecule comprisingsaid first peptide identified in step (a), to identify a second peptidesequence which binds to said molecule and which binds to said antigen ofinterest.
 16. The method of claim 14, in which said first random peptidelibrary is a different library from said second random peptide library.17. The method of claim 14, in which said first random peptide libraryis the same library as said second random peptide library.
 18. Themethod of claim 14 in which the ligand is a receptor.
 19. The method ofclaim 15 in which the antibody is the monoclonal antibody 7E11-C5. 20.The method of claim 14 in which the library of step (a) or step (b) is alibrary of recombinant vectors that express a plurality ofheterofunctional fusion proteins, said fusion proteins comprising abinding domain encoded by an oligonucleotide comprising unpredictablenucleotides in which the unpredictable nucleotides are arranged in oneor more contiguous sequences, wherein the total number of unpredictablenucleotides is greater than or equal to about 15 and less than or equalto about
 600. 21. The method of claim 15 in which the library of step(a) or step (b) is a library of recombinant vectors that express aplurality of heterofunctional fusion proteins, said fusion proteinscomprising a binding domain encoded by an oligonucleotide comprisingunpredictable nucleotides in which the unpredictable nucleotides arearranged in one or more contiguous sequences, wherein the total numberof unpredictable nucleotides is greater than or equal to about 15 andless than or equal to about
 600. 22. The method of claim 14 where thelibrary of step (a) or step (b) is a chemically synthesized library. 23.A method of detecting or measuring an analyte of interest in a sample,comprising: (a) contacting a sample with a molecule comprising a peptidecapable of specifically binding said analyte of interest underconditions such that specific binding between said molecule and saidanalyte can occur; and (b) detecting or measuring the amount of saidbinding in which the presence and amount of said binding indicates thepresence and amount, respectively, of said analyte in the sample; inwhich said peptide is identified by the method of claim
 14. 24. Themethod of claim 23, in which said molecule is immobilized on a solidsubstratum.
 25. A method of determining the location in a patient of atumor comprising: (a) introducing a molecule comprising a peptide thatspecifically binds to a tumor antigen into the patient; and (b)determining the location in the patient of the molecule; in which themolecule is detectably labeled; and in which said peptide is identifiedby a method comprising: (i) screening a first random peptide librarywith an antibody or antigen-binding derivative thereof that specificallybinds to said tumor antigen, to identify a first peptide thatspecifically binds to said antibody or antigen-binding derivativethereof; and (ii) screening a second random peptide library with amolecule comprising said first peptide identified in (i), to identify asecond peptide which binds to said molecule and which binds to saidtumor antigen.
 26. A therapeutic or diagnostic composition comprisingthe molecule of claim 1; and a pharmaceutically acceptable carrier. 27.A therapeutic or diagnostic composition comprising the molecule of claim2; and a pharmaceutically acceptable carrier.
 28. A therapeutic ordiagnostic composition comprising the molecule of claim 5; and apharmaceutically acceptable carrier.
 29. A therapeutic or diagnosticcomposition comprising the molecule of claim 7; and a pharmaceuticallyacceptable carrier.
 30. A therapeutic or diagnostic compositioncomprising the molecule of claim 8; and a pharmaceutically acceptablecarrier.
 31. A therapeutic or diagnostic composition comprising themolecule of claim 12; and a pharmaceutically acceptable carrier.
 32. Acomposition comprising a plurality of molecules of claim 1, in whichsaid peptide sequences of said molecules differ.
 33. A moleculecomprising a peptide or a binding portion thereof which binds to aligand of interest, which peptide is identified by a method comprising:screening a random peptide library with a ligand of interest, saidligand of interest being a peptide having a length of between 5 and 40amino acids, to identify a peptide that specifically binds to the ligandof interest, in which the ligand of interest is also specifically boundby an antibody or a receptor.
 34. The molecule of claim 31 in which theligand is a peptide having a length of between 10 and 20 amino acids.35. A method of obtaining an image of an internal region of a subjectcomprising administering to said subject an effective amount of themolecule of claim 1 in which said molecule is radiolabeled with aradioactive metal, and recording the scintigraphic image obtained fromthe decay of said radioactive metal.
 36. A molecule comprising a peptidewhich binds to a substance of interest, which peptide is identified by amethod comprising: screening a random peptide library with a ligand,said ligand being a peptide of 36 amino acids or fewer, in which theligand is an epitope of an antigen that is specifically bound by anantibody or in which the ligand represents the portion of areceptor-ligand that is responsible for the specific binding of thereceptor to the receptor-ligand.
 37. A peptide comprising the amino acidsequence WQGTHF (SEQ ID NO: 23) and the amino acid sequence LVSKNDSG(SEQ ID NO: 24) that specifically binds to an antigen of human prostatecarcinoma cells.
 38. A molecule comprising an amino acid sequenceselected from the group consisting of: (SEQ ID NO:26)SFMDYFFHTPEPKPAGYPNAYTDPKHPA, (SEQ ID NO:27)SSSIFDYAPFSWGSAGLSNSSINVFERS, (SEQ ID NO:28)SASLWDALGGWTTSAVPSYPRFHQTPGR, (SEQ ID NO:29)SLGLPWIDVFGRSSAEPWPFGRTNLPRS, (SEQ ID NO:30)SVHGAFLDSFFPWAADGPHGRGRLTSF, (SEQ ID NO:31)EEKQGGRWSTMMPRPWCHEGGCGFLYYDAMTKPKTPPIMRTAA, (SEQ ID NO:32)LPRPFDDASWKLRAVKESPDGCGFGSPLLFPPYSGLPTFSSCD, (SEQ ID NO:33)GSFESARGVTCIGNHSIGAHGCGPLRSYASFNRGSGRRH, (SEQ ID NO:34)DQIGSRPQTTSRSISGSWWENAKTLWQQDYAFSAPNAA, (SEQ ID NO:35)LSDAWGNFTTSYRDSAGFPSHAMTTSQGGKRNHASRFP, (SEQ ID NO:36)VQLDDTSPRASGQETSQSEYDARPLLSKFAIPRPWSR, (SEQ ID NO:37)IDSSKNRISGTGYLSFPHIRHANRRHMADDSNLAPGPS, (SEQ ID NO:38)WSIGTHTGPEGKFRIPCDRSGCGGTTLTHGGLNSSPTGQHERP, (SEQ ID NO:39)DPCEDGYWLSSVGRAGASIRGCGAIRRSSRTLTAEYSTRASNH, (SEQ ID NO:40)GSKRSCWGTTISNYFRPVPEGCGSASSINPNTNTGRLPSLHRQ, (SEQ ID NO:41)SSASSGCLGRAEHLDLDSVWGCGSQADMSRRYSPWYGRPRTGV, (SEQ ID NO:42)NVMWSSSKAGIRDCSQVPPGGCGPVNRHRASPPLTPFRHGSIR, (SEQ ID NO:43)PLTSGSSSEYRNRDDCPVYKYATNCPRLNFSPSRYSPF, (SEQ ID NO:44)GDAYGGIFSRPRQGLADSYIHASYTGKHFFRGPRPPTR, (SEQ ID NO:45)STCIGAEGEWKSFHNFLQCRDATSTSSSTLDPTALRFG, (SEQ ID NO:46)YSATLWDQFGSRQVELWSNRHASSALPFASRASVLGSR, (SEQ ID NO:47)ILGWPFLTGLGDSTVHPRGRKGTDPS, (SEQ ID NO:48) SIPSFSMWLNQLGSAALPSKGNSQDRSD,(SEQ ID NO:49) SRDDIFTGGPLVLFRGSKTSNHDVHSMR, (SEQ ID NO:50)RAELVNWYEWFHVTAEAETPVINSHNMT, (SEQ ID NO:51)GAPVWRGNPRWRGPGGFKWPGCGNGPMCNTFTPARGGSRNNGP, (SEQ ID NO:52)GSASSCFPNFTARGVTVGFFGCGSPAHPAAPRVLNPATDFPAP, (SEQ ID NO:53)VFRRTARSSRPIGATVFPWYGCGNSNDETLPHHDSPPSFFLGA, (SEQ ID NO:54)NTCWTDLFWHGLPGGDLPRDGCGLPSELTTHPSRERRDASEN, (SEQ ID NO:55)IDWNWLERGQHNRGYLHSFPDAKSQPTRGPRVAPNGND and (SEQ ID NO:56)GRGSDMREHWPWSMPLILDQHANDPSPRAQSHYYSHPF.


39. A molecule comprising an amino acid sequence selected from the groupconsisting of: (SEQ ID NO:57)VSTGWSGTPRWCAPGGKQGSGCGNGPRWTTLTPDLGGTRKYGP, (SEQ ID NO:57)GAPLWCEKLSGTGSGGFKWPGCGSGPTYNTFTPARVGSDNKWP, (SEQ ID NO:57)GPPVWSAKSRWTGTGVLNWPGCGKVPSCSTYTPSRDRSRKSDP, (SEQ ID NO:57)GSALLTSKGCVRGPGGLMRPGCGNDRLGKSSTYAHGGWIKTGP, (SEQ ID NO:57)GSPVWSGDNRWRGSSPLKRPGCGNGAKCNTLKDNRKDSRKTKH, (SEQ ID NO:57) GPLLPGEAAVHGARGLMRSGCGNGPTWNRLTAACRDSRNKGP, (SEQ ID NO:57)GSPVWMGSTRWTGHGWFRSQGCGNVPRTNSCAPAGKDSQNKGP, (SEQ ID NO:57)GAPVWRGNRWCSDNGELERPGCGYGPRFNILPPGRGNSRKPSP, (SEQ ID NO:57)GSSGWKVKHRCGGPGTLQRPGCGNLPLGHTFPPTRGGSHMEGA, (SEQ ID NO:57)GPRSWMGQPRGSDAGSCKWAGCGDAPMWRASTPGHGGPPNRGS, (SEQ ID NO:57)EALVCRGKPPWSGPAGLLWQGCGTGPVSRTFTSAQGRSRNKTS, (SEQ ID NO:57)GAPVVGDILWCSGARGAKWPGCGKGPTNKTFSHSRGGTQKSGL, (SEQ ID NO:57)GAPVSRCKPACGGFWGVNWPGCGNASMCKTFTNGHGVSSDNGH, (SEQ ID NO:57)GAHGYKNGSTCTGLGGWRCRGCGKGAMCNNPSPAGGAYHNQGP. (SEQ ID NO:57) GPQGSEHQCCSGHWGLKFPGCGNGPICNNFTALRGASRKNGP, (SEQ ID NO:57)GEPVWCRHSGGRVQGGLDWLGCGDGPLRYTVTPARGGPSKHGP, (SEQ ID NO:57)GLSLVRGDSWGSGAGGWKRHGCGHGPMYNPQTPARGGSCTRNT, (SEQ ID NO:57)VSRAWSGKPRLMGSHGLNCPGCGKGHSGIMFIPDPAGSANTPP, (SEQ ID NO:57)CAPMWSGKPPWCVGGGVKFRGCGNRPDCNIITPRLVESRDKAL, and (SEQ ID NO:57)ADPVCSRKPDGGGLRGLRWPGCGKGPILYNVTATTGGSRNNGP.


40. The molecule which binds to a ligand of interest of claim 33 inwhich said ligand comprises VTSAPDTRPAPGSTAPPAHGVTSAPDTR (SEQ ID NO: 9)or a portion thereof.
 41. A therapeutic or diagnostic compositioncomprising a molecule chosen from the group of molecules of claim 38 anda pharmaceutically acceptable carrier.
 42. A therapeutic or diagnosticcomposition comprising a molecule chosen from the group of molecules ofclaim 39 and a pharmaceutically acceptable carrier.
 43. A molecule thatbinds to polymorphic epithelial mucin, comprising an amino acid sequencerepresented by the formula:R₁R₂R₃R₄R₅R₆R₇R₈R₉R₉R₁₀R₁₁R₁₂R₁₃R₁₄R₁₅R₁₆R₁₇R₁₈R₁₉R₂₀R₂₁R₂₂R₂₃R₂₄R₂₅R₂₆(SEQ ID NO: 88) R₂₇R₂₈R₂₉R₃₀R₃₁R₃₂R₃₃R₃₄R₃₅R₃₆R₃₇R₃₈R₃₉R₄₀R₄₁R₄₂R₄₃

wherein: R₁=G, C, E, or V; R₂=A, S, P, or L; R₃=P, T, H, or L; R₄=L, M,Q, G, A, or S; R₅=W or Y; R₆=S, C, K or T; R₇=E, S, C, D, V, or R; R₈=N,H, K, S, or E; R₉=L, H, R, N, Q, T, or G; R₁₀=W, P, R, T, or D; R₁₁=W,C, V, L, or G; R₁₂=S, T, M, or H; R₁₃=G; R₁₄=S, A, G, N, Q, or H; R₁₅=W,H, G, A, or R; R₁₆=G, T, E, P, V, or W; R₁₇=V, F, W, K, or A; R₁₈=K, Q,D, E, R, or L; R₁₉=R, F, or S; R₂₀=P, S, or H; R₂₃=G; R₂₂=C; R₂₃=G;R₂₄=D, S, T, N; R₂₅=G, D, L; R₂₆=P or S; R₂₇=M, S, D, I, L, or R; R₂₈=G,W, C, L, F, Y, or T; R₂₉=S, N, V, F, H, or R; R₃₀=N, A, S, M, or R;R₃₁=F, Q, P, or V; R₃₂=S, V, I, K, A, or S; R₃₃=P, A, N, or Y; R₃₄=G, N,or L; R₃₅=K, R, C, Q or L; R₃₆=V, K, R, or A; R₃₇=G, D, A, or E; R₃₈=S,T, P, Y or W; R₃₉=R, I, L, P, A or S; R₄₀=N, K, or M; R₄₁=S, R, T, E, Q,P, Y or H; R₄₂=G, A, S, D, N, P, Y, or K; R₄₃=P, H or A.
 44. Themolecule of claim 43 wherein: R₁=G; R₂=A; R₃=P; R₅=W; R₆=S; R₁₀=W;R₁₁=w; R₁₂=S or T; R₁₄=S; R₁₆=G; R₁₈=K; R₁₉=R; R₂₀=P; R₂₆=P; R₂₈=G or W;R₃₀=N; R₃₁=F; R₃₃=P; R₃₅=K or R; R₃=G; R₃₈=S; R₄₀=N or K; R₄₂=G;
 45. Themolecule of claim 2 in which the antibody or antigen-binding derivativethereof is capable of specifically binding to a human tumor antigen.